Application of sildenafil in preparation of medicine for improving cell ciliary dysfunction

Abstract

The invention discloses an application of sildenafil in preparation of a medicine for improving cell ciliary dysfunction. According to the application disclosed by the invention, an airway ciliary dysfunction mouse model prepared by a double transgenic mouse for expressing chimeric mouse / human amyloid protein precursor protein (Mo / HuAPP695swe) and mutant human premature protein 1 (PS1-dE9) is jointly treated by hypoxia and a Flk-1 / KDR inhibitor (Semaxanib), and is subjected to intragastric administration with sildenafil; it is verified that sildenafil can relieve the progress of ciliary dysfunction of two model mice, especially improve the functions of lung tissue, cortex and hippocampus cell movement cilia and primary cilia, and a strategy of an action mechanism is provided for understanding that sildenafil serves as a targeted drug for improving ciliary dysfunction.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of sildenafil in the preparation of drugs for improving ciliary dysfunction. Background Art

[0002] The core structure of cell cilia is an axoneme composed of microtubules, and this microtubule structure is the basis of ciliary function. According to different types and positions, it is mainly divided into two categories: motile cilia and primary cilia, with diverse functions, and the specific functions are as follows: Motile cilia generate power through regular swinging, and the main functions include: clearance function, such as the cilia in the respiratory tract swing to push the mucus layer to remove dust, pathogens, and secretions. The cilia in the cerebral ventricles push the cerebrospinal fluid to flow, maintaining intracranial pressure and nutrient transport, etc. Primary cilia do not move and mainly serve as antennas for cell signal transduction, participating in various physiological processes, such as regulating cell proliferation and differentiation through the Hedgehog signaling pathway; participating in embryonic development and tissue homeostasis through the Wnt signaling pathway; regulating cell growth and migration through the DGFRα signaling pathway. Primary cilia disappear during cell division and reform after cell division is completed, and also participate in cell cycle regulation. Ciliary dysfunction can lead to various clinical manifestations, and the specific manifestations depend on the type of cilia and its function in the body. Ciliary dysfunction may lead to various diseases, such as chronic bronchitis, sinusitis, and other pulmonary diseases such as pulmonary vascular diseases in respiratory diseases, brain abnormalities such as hydrocephalus and cognitive dysfunction, and kidney or auditory system diseases.

[0003] Currently, there are some drugs that have been used to treat diseases related to ciliary dysfunction. Sildenafil is a drug mainly used to treat erectile dysfunction and pulmonary arterial hypertension, and its trade name is Viagra. It belongs to a type 5 phosphodiesterase (PDE5) inhibitor. However, there is no report on whether sildenafil has an impact on ciliary dysfunction. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of sildenafil in the preparation of drugs for improving ciliary dysfunction in view of the deficiencies in the prior art.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] Provide the application of sildenafil in the preparation of drugs for improving ciliary dysfunction.

[0007] Furthermore, the drug for improving ciliary dysfunction further includes pharmaceutically acceptable excipients.

[0008] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0009] The airway ciliary dysfunction mouse model of the present invention was prepared by separately treating double transgenic mice expressing chimeric mouse / human amyloid precursor protein (Mo / HuAPP695swe) and mutant human presenilin 1 (PS1-dE9) with hypoxia and the Flk-1 / KDR inhibitor (Semaxanib). Sildenafil was administered by gavage for treatment, and it was verified that sildenafil could alleviate the progression of ciliary dysfunction in the two model mice, especially improving the functions of motile cilia and primary cilia in lung tissue, cortex and hippocampal cells, providing a strategy for the mechanism of action to understand sildenafil as a targeted drug for improving ciliary dysfunction. Brief Description of the Drawings

[0010] Figure 1 Results of Morris water maze and Y maze tests for control group, model group and treatment group mice.

[0011] Figure 2 Shows the enrichment of differentially expressed genes in lung tissue among the control group, model group and treatment group.

[0012] Figure 3 Shows the enrichment of differentially expressed genes in cortical brain tissue among the control group, model group and treatment group.

[0013] Figure 4 Shows the enrichment of differentially expressed genes in hippocampal brain tissue among the control group, model group and treatment group.

[0014] Figure 5 Shows the changes in cilia in lung tissue among the control group, model group and treatment group.

[0015] Figure 6 Shows the changes in the expression of cilia function-related molecules in lung tissue of the control group, model group and treatment group.

[0016] Figure 7 Shows the changes in the expression of cilia function-related molecules in cortical brain tissue of the control group, model group and treatment group. Detailed Embodiments

[0017] The present invention will be further described below in conjunction with the drawings and specific embodiments, but it is not intended to limit the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0018] In the following examples, Student's t-test and analysis of variance were used, and the statistical results were expressed as mean ± standard error (mean ± SE). Each group had at least three samples, and each experiment was repeated at least 3 times. All statistical tests were two-tailed tests, and P < 0.05 was considered statistically significant. SPSS (Statistic Package for Social Science, Chicago, IL, USA) 25.0, GraphPad Prism (San Diego, CA, USA) 8.0, and Image J (National Institutes of Health) 1.8.0 were used for statistical analysis and plotting.

[0019] Unless otherwise specified, the methods in the examples were conventional methods, and the reagents used were conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.

[0020] A mouse model of ciliary dysfunction induced by hypoxia was constructed. The specific construction process and grouping (control group, model group, and treatment group) are as follows: Three-month-old double transgenic mice from Cyagen were purchased. After a single intraperitoneal injection of Flk-1 / KDR inhibitor (20 mg / kg) for one week, they were treated with hypoxia (10% oxygen concentration) for 21 days, and then sildenafil (25 mg / kg / day) was used for treatment for 10 days to prepare a model of multi-cellular ciliary dysfunction in two organs, the lungs and the brain, at a mild degree.

[0021] Example 1

[0022] Detection of mouse lung function: After the treatment ended, 5 mice were randomly selected from each group (control group, model group, and treatment group). The mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital aqueous solution (80 mg / kg), fixed supine on the operating table, the neck skin was longitudinally incised by 1 cm, the subcutaneous tissue was bluntly dissected to expose the trachea, and a "T" incision was made at 2 tracheal rings below the cricoid cartilage. A tracheal cannula was inserted and fixed. The tracheal cannula was connected to a small animal lung function instrument to measure forced vital capacity (FVC), forced expiratory volume in the first 0.1 second (FEV0.1), and peak expiratory flow rate (PEF). Since lung function measurement requires the cooperation of animals, the external pressure method was used to force the mice to inhale deeply and exhale deeply. The mice were placed supine in the airtight body plethysmograph of the respirator. After recording a period of quiet breathing, at the end of exhalation, the syringe was used to quickly inflate through the three-way tube with a volume equivalent to 4 - 6 times the tidal volume (equivalent to deep inhalation), and then immediately disconnected, and negative pressure (-35 cmH2O, 1 cmH2O = 0.098 kPa) was applied to extract air (equivalent to deep exhalation). The volume changes caused were processed by a microcomputer to obtain FVC, FEV0.1, and PEF (Table 1).

[0023] Table 1

[0024]

[0025] From the results in Table 1:

[0026] Compared with the control group, the lung function of rats in the model group was significantly reduced, while that in the treatment group was significantly improved.

[0027] Example 2

[0028] Mouse brain function detection: Detected by Morris water maze (MWM) and Y maze (YM).

[0029] Water maze: Put the mice into the pool and let them swim freely for 1 - 2 minutes to familiarize with the environment. First, conduct hidden platform training, with multiple trainings per day (such as 4 times). Put the mice into the pool from different starting points to find the hidden platform. After finding it, let them stay for 10 - 15 seconds; if not found, guide them to the platform and let them stay for the same time. Then conduct continuous training, usually lasting for 5 - 7 days, and record the time (escape latency) and path for the mice to find the platform. Finally, conduct platform removal test. After the training, remove the platform and let the mice swim freely for 1 minute, and record the staying time and crossing times in the original platform area to evaluate the brain function of memory retention.

[0030] Y maze: Put the mice into the center of the Y maze and let them freely explore for 5 - 10 minutes to familiarize with the environment. Then enter the training stage, let the mice freely explore in the maze, and record the number of times and order of their entering each arm, usually lasting for 5 - 10 minutes. Subsequently, enter the test stage. For the spontaneous alternation test, let the mice freely explore the maze for 5 - 10 minutes, and record the order of their entering each arm to evaluate the changes in spatial working memory and other lung functions.

[0031] As Figure 1 shown, the present invention successfully constructed a mouse model of ciliary dysfunction, and conducted escape latency and alternation times function tests before and after giving sildenafil therapeutic intervention. The experimental results showed that sildenafil could effectively improve or restore the impaired cognitive function.

[0032] Example 3

[0033] After measuring the lung and brain function indexes in this example, samples were taken from peripheral blood, heart, lung tissue and other parts for relevant detections. The specific experimental steps and results are as follows:

[0034] 3.1 Animal specimen collection

[0035] 1) Fix the four limbs of the mice on the dissection table, expose the entire chest and abdomen, cut open the abdomen from bottom to top along the midline of the abdomen until the xiphoid process, turn the liver upwards to expose the porta hepatis, insert the blood collection tube along the direction of the porta hepatis into the portal vein, draw 5 - 10 mL of venous blood, centrifuge at 3000 rmp for 10 minutes at 4℃, take the supernatant, and store it at -80℃ for standby;

[0036] 2) Cut along the costal margins of the mouse bilaterally, lift the xiphoid process to expose the diaphragm, cut open the diaphragm to expose the heart and lungs, irrigate the heart and lung tissues with heparinized saline until the lungs turn white, and then remove the heart and lungs.

[0037] 3) After removing the whole mouse lung tissue, immerse it in physiological saline, take the right upper lung tissue and place it in 4% paraformaldehyde solution for subsequent experiments; the remaining parts are snap-frozen in liquid nitrogen and stored at -80 °C for future use.

[0038] 3.2 Frozen sections of fresh lung tissue

[0039] 1) Immediately after sacrificing the mouse, take fresh lung tissue (1×1×1 cm), and quickly snap-freeze it in liquid nitrogen.

[0040] 2) Apply a layer of OCT embedding medium on the specimen holder, place the snap-frozen tissue above the specimen holder, and put it in a 4 °C refrigerator for 5 - 10 minutes to allow the OCT medium to penetrate the lung tissue.

[0041] 3) Remove the tissue and place it on a glass slide, and freeze the specimen holder.

[0042] 4) Place the tissue above the specimen holder, apply another layer of OCT embedding medium on the tissue, which should completely cover the tissue, and place it on the quick-freezing rack for 30 minutes.

[0043] 5) Cut sections using a cryostat microtome, fix the sections with acetone after cutting, and air-dry them at room temperature.

[0044] 3.3 Paraffin embedding of lung and brain tissues

[0045] 1) Take out the lung and brain tissues fixed with 4% paraformaldehyde solution and place them in a dehydration cassette.

[0046] 2) Dehydration: Place the dehydration cassette in a dehydrator and dehydrate the tissue successively with gradient alcohols, i.e., 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, absolute ethanol for the first dehydration for 30 minutes, and then dehydrate again with fresh absolute ethanol for 30 minutes.

[0047] 3) Clearing: First, treat with 1 / 2 ethanol + 1 / 2 xylene for 60 minutes, then treat with xylene for 60 minutes, and finally replace the xylene and treat for 60 minutes.

[0048] 4) Infiltration: First, treat with 1 / 2 xylene + 1 / 2 paraffin for 90 minutes, then treat with paraffin for 120 minutes, and then replace the paraffin and treat for 120 minutes.

[0049] 5) Embedding: Place the tissue infiltrated with wax in an embedding machine. First, place the melted wax in the embedding frame, take out the tissue and attach a label before the wax solidifies, and cool it on a -20 °C freezing table.

[0050] 6) Sectioning: Place the wax block on a paraffin slicer for sectioning with a thickness of 4 μm. Flatten the tissue, place it on a glass slide, and bake it in an oven at 60 °C until the water is dried and the wax is melted, then take it out and store it at room temperature for later use.

[0051] 3.4 Hematoxylin-eosin (HE) staining

[0052] 1) Deparaffinization to water: Infiltrate the tissue paraffin sections with 100% xylene for deparaffinization three times, 10 minutes each time; then successively through 100% ethanol, 95% ethanol, 75% ethanol, 50% ethanol, and deionized water, 5 minutes for each gradient;

[0053] 2) Staining: Stain with hematoxylin for 5 minutes, and the staining time can be appropriately increased or decreased, then rinse with running water;

[0054] 3) Use a pipette to suck 5% acetic acid and drop it onto the tissue. Differentiate with acetic acid for 1 minute. The color becomes lighter after differentiation, then rinse with running water;

[0055] 4) Stain with eosin for 1 minute, and the staining time can be appropriately increased or decreased, then rinse with running water;

[0056] 5) Dehydration: 70% alcohol, 80% alcohol, 90% alcohol, and absolute alcohol for 1 minute each, and absolute ethanol for 5 minutes;

[0057] 6) Clearing: Treat with xylene for the first time for 2 minutes, and replace the xylene for treatment for 2 minutes;

[0058] 7) Sealing: Cover with a cover slip and seal with neutral gum;

[0059] 8) Image acquisition: Observe the lung and brain tissues under a microscope and take pictures.

[0060] Figure 2 It shows the enrichment of differential genes in lung tissues among the control group, model group, and treatment group, revealing the phenomenon that the ciliary function, microtubules, and axonemes in the model group are significantly inhibited. Specifically, it includes: ciliary components, movement of microtubules, movement of cilia, axoneme components, formation of microtubule bundles, cilia-dependent cell movement, and microtubule transport. And sildenafil can improve the structure and function of these cilia, axonemes, and microtubules.

[0061] Figure 3It shows the enrichment of differentially expressed genes in the cortical brain tissue among the control group, model group, and treatment group. In the model group, six clusters of genes showed abnormal ciliary and microtubule functions, indicating that cilia and microtubules were significantly affected in the model group. After sildenafil treatment, effective regulation of microtubule morphogenesis and formation was observed, meaning that sildenafil can repair ciliary and microtubule dysfunction.

[0062] Figure 4 It shows the enrichment of differentially expressed genes in the hippocampal brain tissue among the control group, model group, and treatment group. In the model group, five clusters of genes showed inhibition of functions such as microtubule motility, ciliary organization, and microtubule transport, indicating that microtubules and cilia were significantly affected in the model group. After sildenafil treatment, effective improvement of these functions was observed, meaning that sildenafil can repair microtubule and ciliary dysfunction and restore normal cell structure and function.

[0063] Figure 5 It shows the changes in lung tissue cilia among the control group, model group, and treatment group. The results show that in the model group, the number of cilia in mice not only decreased, but also the length was significantly shortened, indicating that cilia were significantly damaged. After sildenafil treatment, both the number and length of cilia were significantly improved, suggesting that sildenafil has the ability to repair ciliary damage.

[0064] Figure 6 It shows the changes in the expression of cilia function-related molecules in the lung tissue of the control group, model group, and treatment group. The results show that the expression of DNH5, HYDIN, FOXJ1, and DNAH10 in the model group was lower than that in the control group. After sildenafil treatment, the expression of these cilia function-related molecules was significantly upregulated, suggesting that sildenafil can effectively improve ciliary damage and restore its normal function.

[0065] Figure 7 It shows the changes in the expression of cilia function-related molecules in the cortical brain tissue of the control group, model group, and treatment group. The results show that the expression of DNH5, HYDIN, FOXJ1, and DNAH10 in the model group was lower than that in the control group. After sildenafil treatment, the expression of these cilia function-related molecules was significantly upregulated, suggesting that sildenafil can effectively improve ciliary damage and restore its normal function.

[0066] The above are only preferred embodiments of the present invention, and do not limit the implementation mode and protection scope of the present invention accordingly. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the content and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of sildenafil in the preparation of a drug for improving ciliary dysfunction of cells.

2. The application according to claim 1, wherein The drug for improving ciliary dysfunction of cells further comprises a pharmaceutically acceptable excipient.

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