Application of rapamycin in relieving slow obstructive pulmonary disease caused by down-regulation or deletion of peroxidase 6

Abstract

The invention discloses application of rapamycin in relieving PRDX6 down-regulation or deficiency chronic obstructive pulmonary disease, and belongs to the technical field of biological medicine. After mouse alveolar epithelial cells PRDX6 are knocked out, emphysema phenotype aggravation caused by elastinase airway instillation is found, and emphysema injury can be treated and improved by rapamycin. The invention relates to a novel application of rapamycin, which is characterized in that an immunosuppressant rapamycin in clinical medicine is used for treating chronic obstructive pulmonary disease caused by PRDX6 down-regulation or deficiency for the first time, and the application has important significance in the aspects of expanding indications of rapamycin, reducing the drug development cost and the like. And a research direction is provided for follow-up research on chronic obstructive pulmonary disease pathogenesis and development of novel treatment drugs.

Description

Technical Field

[0001] The present invention relates to the application of rapamycin in alleviating chronic obstructive pulmonary disease (COPD) with down - regulation or deletion of peroxiredoxin 6 (PRDX6), belonging to the field of biomedical technology. Background Art

[0002] COPD is a disease mainly characterized by chronic airway inflammation and emphysema. Currently, the standard use of long - acting bronchodilators and inhaled corticosteroids can only partially relieve clinical symptoms, and it is still difficult to improve lung function and delay the disease progression of COPD.

[0003] PRDX6 is an important antioxidant enzyme involved in intracellular redox reactions. Regarding the role of PRDX6 in COPD, some studies have shown that the polymorphism of the PRDX6 gene is related to the risk of COPD in the Han Chinese population. In an in vitro model stimulated by cigarette smoke extract, the expression level of PRDX6 decreases, and it is negatively correlated with the expression levels of cell senescence markers - cyclin - dependent kinase inhibitors p16 and p21. Previously, we also detected the content of PRDX6 in the lung tissues of COPD patients and epithelial cells stimulated by cigarette smoke extract and found that the expression of this protein was down - regulated; over - expression of PRDX6 can reduce cell death caused by cigarette smoke extract stimulation. The above research results suggest that PRDX6 may play a protective role in the pathogenesis of COPD.

[0004] By deeply studying the role of PRDX6 in COPD, it may provide new targets and strategies for the treatment of this disease. However, it still takes time to develop a targeted activator of PRDX6. Therefore, if a small - molecule drug that can effectively relieve the clinical symptoms of COPD and delay the disease progression is found, it is expected to achieve precise treatment for COPD patients with decreased or absent PRDX6 caused by environmental factors such as smoking and atmospheric particulate matter. Summary of the Invention

[0005] The object of the present invention is to provide the application of rapamycin in alleviating COPD in mice caused by the deletion of peroxiredoxin 6.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the application of rapamycin in the preparation of a drug for preventing and / or treating COPD, wherein the COPD is COPD with down - regulation or deletion of PRDX6 in lung tissue.

[0008] Preferably, the COPD with down - regulation or deletion of PRDX6 includes COPD caused by environmental factors such as smoking and atmospheric particulate matter.

[0009] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient is rapamycin.

[0010] Preferably, the dosage form of the drug is an injection, a tablet, a powder, or a capsule.

[0011] The present invention also provides the use of rapamycin in the preparation of a drug for preventing and / or treating emphysema, wherein the PRDX6 in the emphysema lung tissue is down-regulated or absent.

[0012] The present invention also provides the use of a mouse model with Prdx6 gene knockout in alveolar epithelial cells in the screening of a drug for preventing and / or treating chronic obstructive pulmonary disease (COPD), wherein the COPD is a COPD with down-regulation or absence of PRDX6 in the lung tissue.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention for the first time uses rapamycin as a therapeutic drug, and in vivo experiments in animals show that rapamycin has a good therapeutic effect on emphysema in mice exacerbated by the decrease of PRDX6; the present invention solves the problem of the long cycle of developing a PRDX6-targeted activator; at the same time, it also solves the problem that current drugs for treating COPD only control clinical symptoms and cannot delay the progression of the disease. Rapamycin provided by the present invention is expected to provide a precise drug treatment plan for COPD patients with decreased or absent PRDX6. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a result display of rapamycin improving emphysema in Prdx6 knockout mice in alveolar epithelial cells. Among them, A is a HE staining map of lung tissue; B is the statistical result of mouse body weight injury; C is the statistical result of the average alveolar septum; D is the statistical result of lung volume; Rapamycin did not improve the following indicators (lung tissue destruction, body weight injury, average alveolar septum, and lung volume) in wild group mice; but improved the above indicators in Prdx6 knockout mice; ns indicates no significant statistical difference, *p < 0.05, **p < 0.01, ****p < 0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] To make the present invention more obvious and understandable, preferred embodiments are described in detail below in conjunction with the accompanying drawings.

[0017] In the present invention, by discovering the expression changes of PRDX6 in clinical samples of chronic obstructive pulmonary disease (COPD) and the specific mechanism by which PRDX6 affects the development of COPD, it is proposed for the first time to use rapamycin as a therapeutic drug. Animal experiments in vivo have shown that rapamycin can improve the COPD emphysema in mice caused by the knockout of Prdx6 in alveolar epithelial cells. In wild-type mice, after intratracheal instillation of elastase, the body weight of the mice decreased; the pathological results of lung tissues indicated alveolar destruction, an increased average alveolar distance, and an enlarged lung volume. After rapamycin treatment, the above indicators reflecting emphysema did not improve. Although the emphysema injury caused by intratracheal instillation of elastase was more severe in Prdx6 knockout mice of alveolar epithelial cells than in wild-type mice, in Prdx6 knockout mice of alveolar epithelial cells, the rapamycin treatment group had less weight loss, a shorter average alveolar distance, and a smaller lung volume compared with the simple model group. Rapamycin treatment made the emphysema condition of Prdx6-deficient mice in alveolar epithelial cells not significantly different from that of wild-type mice. Therefore, rapamycin can improve the severity of emphysema caused by Prdx6 deficiency.

[0018] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. The materials, reagents, etc. used in the following examples are, unless otherwise specified, reagents and materials obtained from commercial sources.

[0019] The experimental materials involved in the present invention are as follows:

[0020] 1. Rapamycin was purchased from Selleck, and its structural formula is shown as follows:

[0021]

[0022] 2. Porcine pancreatic elastase was purchased from Sigma-Aldrich, E7885.

[0023] 3. Inducible Prdx6 gene knockout mice of alveolar epithelial cells were purchased from Southern Model Animal Co., Ltd. Inducible means that the specific knockout of the Prdx6 gene in alveolar epithelial cells is achieved by inducing the activation of the CreER system through tetracycline induction (Tet-on / off) or tamoxifen.

[0024] Example:

[0025] Wild-type and alveolar epithelial cell Prdx6 gene knockout mice were randomly divided into a control group, a model group, and a rapamycin treatment group (male, 8 - 10 weeks old, 22 - 25 g per mouse, 5 mice per group). In the emphysema model group and the rapamycin treatment group of mice, porcine pancreatic elastase (31.25 μg - 50 μl / mouse / time) was instilled into the airway, and an equal volume of normal saline was instilled into the airway of the control group. From the 8th day after modeling, the rapamycin treatment group was intraperitoneally injected with 1 mg / kg of rapamycin once a day for seven consecutive days. After 24 hours of the last injection, the mice were weighed, sacrificed after performing chest CT imaging of the mice, and lung tissue samples were collected for HE staining and pathological analysis, and the body weight injury of the mice was detected. The entire modeling cycle was 15 days.

[0026] 2. Chest CT Imaging of Mice

[0027] (1) Anesthesia protocol: Induction with 3% isoflurane and maintenance with 1.5 - 2% (oxygen flow rate 1 L / min);

[0028] (2) Position: Fixed in the prone position on a dedicated animal bed, keeping the chest wall naturally extended;

[0029] (3) CT imaging scan: Using X-rays with 55 kV and 145 μA, a receiver with 2048×2048 pixels, 360° rotational scanning, collecting 1 projection image every 0.4°, and the collection time was about 6 minutes;

[0030] (4) Image post-processing: Three-dimensional reconstruction of the lungs, and the analysis area ranged from the upper part of the mouse chest to the starting part of the tracheal bifurcation and down to the middle part of the liver to ensure that all lung tissue was included. The lung volume was calculated.

[0031] 3. Hematoxylin-Eosin (HE) Staining of Lung Tissue

[0032] (1) Fixation and sampling: Immediately after the mouse lung tissue was removed, it was immersed in 4% paraformaldehyde at room temperature for fixation overnight, and the tissue was placed in an embedding frame and labeled;

[0033] (2) Dehydration and wax infiltration: Dehydration with gradient alcohol, 75% alcohol for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h; absolute ethanol I and II for 30 min each; alcohol-benzene for 5 - 10 min, xylene I and II for 5 - 10 min each; paraffin I, II, and III melted at 65°C for 1 h each;

[0034] (3) Paraffin embedding and sectioning: Melting the wax and putting the tissue into the embedding frame, cooling, and waiting for the wax to solidify;

[0035] (4) Paraffin sectioning: Sectioning the wax block, 4 μm thick, placing it on a glass slide, and baking it in an oven at 60°C;

[0036] (5) Dewax the paraffin section to water: Treat it successively with dewaxing solution, absolute ethanol, and 75% alcohol. Treat it with the pretreatment solution for 1 min;

[0037] (6) Hematoxylin staining: Stain with hematoxylin staining solution for 5 minutes, rinse with water, differentiate with the differentiating solution, rinse with water again, and finally blue the section with the bluing solution and rinse with water again;

[0038] (7) Eosin staining: First dehydrate with 95% ethanol for 1 minute, and then stain with eosin for 15 seconds;

[0039] (8) Dehydrate, mount the slide and observe under the microscope.

[0040] The experimental results are as Figure 1 shown. Compared with the simple model group, the lung tissue injury in the rapamycin treatment group was significantly improved ( Figure 1 A), the weight loss was reduced ( Figure 1 B), the average alveolar distance was shortened ( Figure 1 C), and the lung volume was decreased ( Figure 1 D). Rapamycin treatment made the emphysema condition of Prdx6-deficient alveolar epithelial cell mice show no significant difference compared with wild-type mice. Therefore, rapamycin can improve the severity of emphysema caused by Prdx6 deficiency.

[0041] Therefore, for patients clinically diagnosed with chronic obstructive pulmonary disease (COPD) and whose lung tissue samples are detected with down-regulated Prdx6 gene or protein expression, rapamycin or a combination of rapamycin and other COPD treatment drugs can be administered for treatment.

[0042] The above is only the preferred embodiment of the present invention, and it does not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of rapamycin in the preparation of a medicament for preventing and / or treating chronic obstructive pulmonary disease, characterized in that, The chronic obstructive pulmonary disease (COPD) shown is a COPD with downregulation or deletion of PRDX6 in lung tissue.

2. The application according to claim 1, wherein The COPD with downregulation or deletion of PRDX6 includes COPD caused by smoking or environmental factors.

3. The application according to claim 1, characterized in that, The drug includes an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient is rapamycin.

4. The application according to any one of claims 1 to 3, characterized in that The dosage form of the drug is an injection, tablet, powder, or capsule.

5. Use of rapamycin in the preparation of a medicament for preventing and / or treating emphysema, characterized in that, The emphysema shown is downregulation or deletion of PRDX6 in lung tissue.

6. Use of a mouse model with Prdx6 gene knockout in alveolar epithelial cells in screening for drugs for preventing and / or treating chronic obstructive pulmonary disease, characterized in that, The COPD is a COPD with downregulation or deletion of PRDX6 in lung tissue.

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