Application of NOP2 expression inhibitor in preparation of medicine for preventing and treating chronic obstructive pulmonary disease

Abstract

The invention discloses application of an NOP2 expression inhibitor in preparation of a medicine for preventing and treating chronic obstructive pulmonary disease. It is found that by inhibiting expression of targeted NOP2, lung tissue damage of chronic obstructive pulmonary disease model mice can be effectively improved, lung tissue inflammatory response is inhibited, and lung functions are improved. Therefore, the NOP2 expression inhibitor has the prospect of being developed into the medicine for preventing or treating the chronic obstructive pulmonary disease, and the NOP2 expression inhibitor is nucleic acid (such as RNA interference fragment shRNA, siRNA, miRNA, dsRNA and the like of targeted NOP2) for inhibiting NOP2 expression or a carrier (such as a plasmid carrier and a virus carrier) capable of releasing or expressing the nucleic acid.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the application of inhibitors of known targets in pharmaceutical manufacturing, specifically the application of NOP2 expression inhibitors in the preparation of drugs for the prevention and treatment of chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by persistent airflow limitation. Cigarette smoke, toxic particulate matter, and gases are the main risk factors for COPD. Current treatments for COPD mainly include bronchodilators, corticosteroids, and protease inhibitors. While these can effectively alleviate clinical symptoms, they are not effective in inhibiting the progressive deterioration of lung function and the continued progression of the disease.

[0003] The human NOP2 gene is located in the 12p13.31 region of chromosome 12. This gene is widely expressed in various human tissues, including lymph nodes and bone marrow, and its expression can be detected in more than 25 different tissues, with subcellular localization concentrated in the nucleolar region. During the cell cycle, its expression occurs in the G1 phase, peaking in the early S phase. Current research has found that abnormal NOP2 expression is often associated with tumors. For example, in gastric adenocarcinoma, upregulated NOP2 expression promotes tumor growth and predicts a poor prognosis; in hepatocellular carcinoma, it is associated with cancer progression by mediating m5C methylation of XPD. These characteristics make it a promising biomarker for tumor diagnosis and prognosis, and also provide direction for targeted therapy research on related tumors.

[0004] There are currently no reports on the relationship between NOP2 and the prevention and treatment of chronic obstructive pulmonary disease, hence this invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of NOP2 expression inhibitors in the preparation of drugs for the prevention and treatment of chronic obstructive pulmonary disease.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] Application of NOP2 expression inhibitors in the preparation of drugs for the prevention or treatment of chronic obstructive pulmonary disease.

[0008] Preferably, the NOP2 expression inhibitor is a nucleic acid that inhibits NOP2 expression, or a vector that can release or express the nucleic acid.

[0009] More preferably, the nucleic acid that inhibits NOP2 expression is an RNA interference fragment targeting NOP2. In a specific embodiment, a shRNA (NOP2-shRNA) targeting the NOP2 gene is provided. Those skilled in the art know that shRNA is a commonly used RNA interference tool, and its core mechanism of action is to silence gene expression at the posttranscriptional level.

[0010] More preferably, the RNA interference fragment targeting NOP2 is selected from one or more of shRNA, siRNA, miRNA, and dsRNA. Those skilled in the art know that after shRNA enters the cell, it is cleaved by an enzyme called "Dicer," removing the stem-loop structure to form mature siRNA that interferes with the target gene. Of course, those skilled in the art can also choose RNA interference tools such as miRNA and dsRNA.

[0011] Preferably, the vector is a plasmid vector or a viral vector.

[0012] More preferably, the viral vector is selected from one of lentiviral vectors, adeno-associated virus vectors, and adenovirus vectors.

[0013] More preferably, the drug uses an NOP2 expression inhibitor as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0014] More preferably, the excipient is a liquid, solid, or semi-solid excipient.

[0015] More preferably, the dosage form is selected from one of the following: injection, microneedle, lyophilized powder, nebulized liquid, tablet, capsule, emulsion, nasal drops, spray, and aerosol.

[0016] Beneficial effects:

[0017] This invention reveals that targeting and inhibiting NOP2 expression can effectively improve lung tissue damage, suppress pulmonary inflammation, and enhance lung function in a mouse model of chronic obstructive pulmonary disease (COPD). Therefore, NOP2 expression inhibitors hold promise for development into drugs for the prevention or treatment of COPD. These inhibitors may be nucleic acids that inhibit NOP2 expression (such as NOP2-targeting RNA interference fragments like shRNA, siRNA, miRNA, and dsRNA) or vectors that release or express this nucleic acid (such as plasmid vectors or viral vectors). Attached Figure Description

[0018] Figure 1 The mRNA levels of NOP2 in the lung tissues of mice in each group; among them: compared with the NC-ShRNA blank group, a p < 0.05 aap < 0.01; compared with the NC-ShRNA COPD group, c p < 0.05 cc p < 0.01;

[0019] Figure 2 HE staining was used to observe the airways and alveoli of mice in each group;

[0020] Figure 3 The number and diameter of alveoli in the lung tissue of mice in each group were measured; among them, compared with the NC-ShRNA blank group, a p < 0.05 aa p < 0.01; compared with the NC-ShRNA COPD group, c p < 0.05 cc p < 0.01;

[0021] Figure 4 The lung function levels (TV, MV, PEF, EF50) of mice in each group were measured; among them, compared with the NC-ShRNA blank group, a p < 0.05 aa p < 0.01; compared with the NC-ShRNA COPD group, c p < 0.05 cc p < 0.01;

[0022] Figure 5 The levels of IL-1β, IL-6, and TNF-α in the lung tissue of mice in each group; among them, compared with the NC-ShRNA blank group, a p < 0.05 aa p < 0.01; compared with the NC-ShRNA COPD group, c p < 0.05 cc p < 0.01;

[0023] Figure 6 The levels of antioxidant proteins, matrix metalloproteinases, and mucins in the lung tissue of mice in each group were measured; among them, compared with the NC-ShRNA blank group, a p < 0.05 aa p < 0.01; compared with the NC-ShRNA COPD group, c p < 0.05 cc p < 0.01. Detailed Implementation

[0024] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0025] I. Experimental Materials

[0026] Twenty-four C57BL / 6J mice (male, 19 - 21 g) were purchased from Beijing Speyford Biotechnology Co., Ltd., with the license number SYXK(Yu) 2021 - 0015.

[0027] Hongqiqu filter tip cigarettes (flue-cured type, tar content 10 mg, nicotine content 0.8 mg, nitric oxide 12 mg) were produced by China Tobacco Henan Industrial Co., Ltd.

[0028] Adeno-associated virus 6 (AAV6) includes negative control shRNA (NegativeControl shRNA, NC ShRNA) and NOP2-shRNA wrapped by mir30 flanks driven by CMV promoter. It was designed, synthesized and packaged into a virus solution with a virus titer of 10 13 VG / mL by Shanghai Anzhen Biotechnology Co., Ltd. The transcript of mouse NOP2 is: NM_138747.2.

[0029] Mouse Interleukin-1β (IL-1β) ELISA Kit (DY401) was purchased from R&D Systems; IL-6 ELISA Kit (555240), mouse Tumor necrosis factor-α (TNF-α) were purchased from BD; Total Superoxide Dismutase (T-SOD) activity detection kit (E-BC-K020-M) was purchased from Elabscience; Mouse Matrix Metalloproteinase12 (MMP-12) ELISA Kit (RE1068M) was purchased from Reed Biotech; Mouse Mucin-5 subtypeAC (MUC5AC) ELISA Kit (EM1220) was purchased from Fine Test.

[0030] II. Experimental methods

[0031] 1. Establishment and grouping of animal models

[0032] C57BL / 6J mice were randomly divided into NC-ShRNA blank group, NC-ShRNA COPD group, NOP2-ShRNA blank group, and NOP2-ShRNA COPD group, with 6 mice in each group.

[0033] The NC-ShRNA COPD group and the NOP2-ShRNA COPD group established a COPD mouse model through repeated cigarette smoke exposure from week 1 to week 12 (1-8 cigarettes were given for adaptive exposure each time from week 1 to week 2, and 8 cigarettes were given each time from week 2 to week 12, 6 days a week, twice a day, 60 min each time, with a 3 h interval between each exposure). The NOP2-ShRNA blank group and the NOP2-ShRNA COPD group were given 50 μL / mouse of NOP2-shRNA (1×10⁻⁶) via intratracheal infusion at weeks 6 and 9. 12 VG / mL), the NC-ShRNA blank group and the NC-ShRNA COPD group were administered 50 μL / animal NC-shRNA (1×10) via tracheal infusion at weeks 6 and 9. 12 VG / mL), with follow-up tests conducted after the second exposure at the end of week 12.

[0034] 2. Lung function test

[0035] After 12 weeks of exposure, mice were euthanized using the WBP small animal whole-body volume plethysmography system to measure tidal volume (TV), minute ventilation volume (MV), 50% tidal volume expiratory flow (EF50), and peak expiratory flow (PEF). Lung function tests were also performed.

[0036] 3. Preparation of bronchoalveolar lavage fluid

[0037] The mouse neck skin was opened to expose the trachea and an incision was made. A flat-tipped needle was inserted into the tracheal incision, and 1 mL of PBS buffer was injected into the syringe to irrigate the mouse lung tissue. The above irrigation operation was repeated 3 times. The irrigation fluid was combined and centrifuged, and the supernatant was collected for later use.

[0038] 4. Pathological observation of lung tissue

[0039] The left lung was instilled with formaldehyde and then fixed. After 72 hours, it was embedded in paraffin and stained with hematoxylin and eosin (HE). The alveoli and airways were photographed at 200× using CaseViewer software.

[0040] 5. ELISA detection of inflammatory factors, matrix metalloproteinases, and mucin levels in lung tissue.

[0041] The expression levels of IL-1β and MMP12 in mouse lung tissue were detected by homogenizing the right lung tissue; the expression levels of IL-6, TNF-α, and MUC5AC were detected by bronchoalveolar lavage fluid of mice; and the activity of T-SOD in mouse bronchoalveolar lavage fluid was detected by colorimetric method.

[0042] III. Statistical Analysis

[0043] Statistical analysis was performed using SPSS 23.0 software, employing one-way ANOVA. LSD-t method was used for data with homogeneous variances, and Dunnett's T3 method was used for data with unequal variances. Data are expressed as mean ± standard deviation (mean ± SD). A p-value < 0.05 was considered statistically significant.

[0044] IV. Experimental Results

[0045] 1. Comparison of NOP2 expression levels among groups

[0046] like Figure 1 As shown, compared with the NC-ShRNA blank group, the NOP2 mRNA level in the lung tissue of mice in the NC-ShRNA COPD group was significantly increased (indicating that COPD upregulates NOP2 expression), while the NOP2 mRNA level in the lung tissue of mice in the NOP2-ShRNA blank group was significantly decreased (indicating that NOP2-ShRNA achieved the expected downregulation of NOP2 expression); compared with the NC-ShRNA COPD group, the NOP2 mRNA level in the lung tissue of mice in the NOP2-ShRNA COPD group was significantly decreased (again indicating that NOP2-ShRNA achieved the expected downregulation of NOP2 expression); compared with the NOP2-ShRNA blank group, there was no significant change in the NOP2 mRNA level in the lung tissue of the NOP2-ShRNA COPD group.

[0047] 2. NOP2-ShRNA improves lung tissue pathology in COPD mice

[0048] Results of HE staining of lung tissue as follows Figure 2 As shown in the figure, the lung tissue structure of mice in the NC-ShRNA blank group was normal, with alveoli of regular size and arrangement, and no obvious inflammatory cell infiltration was observed. Compared with the NC-ShRNA blank group, the NC-ShRNA COPD group showed obvious inflammatory cell infiltration, increased alveolar structural breakage, and alveolar wall thickening, indicating that the above modeling method can achieve the expected modeling effect. The lung tissue structure of mice in the NOP2-ShRNA blank group and the NOP2-ShRNA COPD group was normal, with alveoli of regular size and arrangement, and no obvious inflammatory cell infiltration was observed, which was significantly better than the NC-ShRNA COPD group. In particular, the comparison between the NOP2-ShRNA COPD group and the NC-ShRNA COPD group shows that downregulating NOP2 expression can effectively inhibit the lung damage caused by COPD modeling.

[0049] Pathological scoring results as follows Figure 3As shown in the figure. Compared with the NC-ShRNA control group, the average alveolar diameter of mice in the NC-ShRNA COPD group was significantly increased, and the number of alveoli per unit area was significantly reduced, indicating that the above modeling method can achieve the expected modeling effect. Compared with the NOP2-ShRNA control group, there were no significant changes in the average alveolar diameter and the number of alveoli per unit area in the NOP2-ShRNA COPD group. Compared with the NC-ShRNA COPD group, the average alveolar diameter of mice in the NOP2-ShRNA COPD group was significantly decreased, and the number of alveoli per unit area was significantly increased, indicating that downregulating NOP2 expression can effectively inhibit the damage to lung tissue caused by COPD modeling.

[0050] 3. NOP2-ShRNA improves lung function in COPD mice

[0051] like Figure 4 As shown, compared with the NC-ShRNA blank group, the TV, MV, PEF, and EF50 of mice in the NC-ShRNA COPD group were significantly reduced, indicating that the above modeling method can achieve the expected modeling effect; compared with the NOP2-ShRNA blank group, there were no significant changes in TV, MV, PEF, and EF50 in the NOP2-ShRNA COPD group; compared with the NC-ShRNA COPD group, TV, MV, PEF, and EF50 of the NOP2-ShRNA COPD group were significantly increased, indicating that downregulating NOP2 expression can effectively improve lung function in COPD mice.

[0052] 4. NOP2-ShRNA improves lung tissue inflammatory response in COPD mice

[0053] like Figure 5 As shown, compared with the NC-ShRNA blank group, the levels of IL-1β, IL-6, and TNF-α in the lung tissue of mice in the NC-ShRNA COPD group were significantly increased, indicating that the above modeling method can achieve the expected modeling effect. Compared with the NOP2-ShRNA blank group, the levels of IL-1β, IL-6, and TNF-α in the lung tissue of mice in the NOP2-ShRNA COPD group were not significantly changed. Compared with the NC-ShRNA COPD group, the levels of IL-1β, IL-6, and TNF-α in the lung tissue of mice in the NOP2-ShRNA COPD group were significantly decreased, indicating that downregulating NOP2 expression can effectively inhibit the inflammatory response in lung tissue caused by COPD modeling.

[0054] 5. NOP2-ShRNA inhibits oxidative stress, increased matrix metalloproteinases, and hypersecretion of mucus in COPD mice.

[0055] like Figure 6As shown, compared with the NC-ShRNA blank group, the activity of superoxide dismutase (SOD) in the lung tissue of mice in the NC-ShRNA COPD group was significantly reduced, while the levels of matrix metalloproteinase (MMP-12) and mucin (MUC5AC) were significantly increased, indicating that the above modeling method can achieve the expected modeling effect. Compared with the NC-ShRNA COPD group, the activity of superoxide dismutase (SOD) in the lung tissue of mice in the NOP2-ShRNA COPD group was significantly increased, while the levels of matrix metalloproteinase (MMP-12) and mucin (MUC5AC) were significantly reduced. SOD is an antioxidant metalloenzyme present in organisms, which can inhibit inflammation through its antioxidant effect. MMP-12 is secreted by inflammatory cells such as macrophages and plays a key role in diseases such as COPD; its overexpression may lead to pathological damage. Mucin (MUC5AC) has a promoting effect on airway inflammation in COPD. These results indicate that downregulating NOP2 expression may exert its inhibitory effect on tissue damage and improve lung function in COPD model mice by increasing superoxide dismutase (SOD) activity and decreasing the levels of matrix metalloproteinase (MMP-12) and mucin (MUC5AC).

[0056] In summary, targeting and inhibiting NOP2 expression effectively improves lung tissue damage, suppresses pulmonary inflammation, and enhances lung function in a mouse model of chronic obstructive pulmonary disease (COPD). Therefore, NOP2 expression inhibitors hold promise for development into drugs for the prevention or treatment of COPD. These inhibitors could be nucleic acids that inhibit NOP2 expression (such as NOP2-targeting RNA interference fragments like shRNA, siRNA, miRNA, and dsRNA) or vectors that release or express this nucleic acid (such as plasmid vectors or viral vectors).

[0057] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Application of NOP2 expression inhibitors in the preparation of drugs for the prevention or treatment of chronic obstructive pulmonary disease.

2. In the application according to claim 1, the NOP2 expression inhibitor is a nucleic acid that inhibits NOP2 expression, or a vector that can release or express the nucleic acid.

3. In the application according to claim 2, the nucleic acid that inhibits NOP2 expression is an RNA interference fragment that targets NOP2.

4. In the application according to claim 3, the RNA interference fragment targeting NOP2 is selected from one or more of shRNA, siRNA, miRNA, and dsRNA.

5. The application according to claim 2, wherein the vector is a plasmid vector or a viral vector.

6. In the application according to claim 5, the viral vector is selected from one of lentiviral vectors, adeno-associated virus vectors, and adenovirus vectors.

7. The application according to any one of claims 1 to 6, wherein the drug is formulated into a pharmaceutically acceptable dosage form using an NOP2 expression inhibitor as the active ingredient and pharmaceutically acceptable excipients.

8. The application according to claim 7, wherein the excipient is a liquid, solid, or semi-solid excipient.

9. The application according to claim 7, wherein the dosage form is selected from one of injections, microneedles, lyophilized powders, nebulized liquids, tablets, capsules, emulsions, nasal drops, sprays, and aerosols.

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