Application of nicotinamide adenine dinucleotide in preparation of medicine for preventing and / or treating primary stationary cilia syndrome

Abstract

The invention provides application of nicotinamide adenine dinucleotide in preparation of a medicine for preventing and / or treating primary stationary cilia syndrome, and belongs to the field of medicine. It is found for the first time that nicotinamide adenine dinucleotide can effectively recover the ciliary movement function, and the nicotinamide adenine dinucleotide has a wide application prospect in preparation of the medicine for preventing and / or treating the primary fixed ciliary syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to the use of nicotinamide adenine dinucleotide in preparing a medicine for preventing and / or treating primary immotile cilia syndrome. Background Art

[0002] Primary ciliary dyskinesia (PCD), also known as immobile ciliary syndrome, primary ciliary dyskinesia, primary immobile ciliary syndrome or primary immobile ciliary syndrome, is a hereditary disease. PCD is mainly caused by gene mutations related to ciliary movement. These mutations can lead to defects in the ultrastructure of cilia, which in turn affect the normal movement function of cilia. The treatment of PCD is single, and there is no symptomatic drug. It usually uses treatment methods similar to cystic fibrosis, but the pathogenesis of cystic fibrosis is completely different from PCD. Conservative medical treatment is the basis of PCD treatment. The key is to strengthen airway management and conduct regular respiratory function assessment. This includes keeping the airway open, avoiding infection, and providing necessary nutritional support and care. Physical therapy plays an important role in the treatment of PCD, mainly including: Postural drainage: by changing the patient's position, using gravity to promote sputum discharge. Patting the back to expectorate: by patting the patient's back, the sputum is loosened and discharged. Respiratory function exercise: by performing specific respiratory exercises, the strength of respiratory muscles is enhanced and respiratory function is improved. Drug treatment mainly uses antibiotics and mucus promoters to achieve anti-inflammation and promote sputum discharge, thereby alleviating respiratory symptoms. Currently, there are no drugs for the clinical treatment of PCD that can restore ciliary function.

[0003] Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme widely present in organisms. It plays a vital role in cells and participates in regulating a variety of biochemical reactions. NAD+ can be synthesized from tryptophan and vitamin B3 (niacin and nicotinamide). The intermediates nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) can also promote NAD+ synthesis. Energy metabolism: NAD+ is a key substance connecting the tricarboxylic acid cycle and the respiratory chain, and plays an irreplaceable role in glycolysis, gluconeogenesis, the tricarboxylic acid cycle and the respiratory chain. As a carrier of hydrogen, it synthesizes ATP in the respiratory chain through chemical osmotic coupling to provide energy for cells. Studies have shown that NAD+ participates in the DNA repair process and helps maintain the stability of the genome. NAD+ plays an important role in the nervous system and aging-related diseases, and can be used to treat diseases such as Alzheimer's disease, Parkinson's disease and depression. In addition, a decrease in NAD+ levels is a sign of aging, and increasing NAD+ levels can slow down the aging process. NAD+ acts as an antioxidant, helping to reduce signs of aging and is an important element in skin care. Importantly, by enhancing NAD+ levels, the secretion of inflammatory cytokines such as IL-1β and TNF-α can be reduced, thereby exerting an anti-inflammatory effect. There are currently no reports of nicotinamide adenine dinucleotide being used to treat primary ciliary immobility syndrome. Summary of the invention

[0004] The object of the present invention is to provide use of nicotinamide adenine dinucleotide in preparing a drug for preventing and / or treating primary immotile cilia syndrome.

[0005] The present invention provides use of nicotinamide adenine dinucleotide in preparing a medicine for preventing and / or treating primary immotile cilia syndrome.

[0006] Furthermore, the drug is a drug that restores ciliary movement function.

[0007] Furthermore, the drug is a pharmaceutical preparation prepared with nicotinamide adenine dinucleotide as an active ingredient and pharmaceutically acceptable excipients.

[0008] Furthermore, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0009] Furthermore, the oral preparation is a tablet, a granule, or a capsule.

[0010] Furthermore, the injection preparation is a powder injection.

[0011] The present invention discovers for the first time that nicotinamide adenine dinucleotide can effectively restore the movement function of cilia, and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile cilia syndrome.

[0012] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0013] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the research of the present invention.

[0015] Figure 2 PCD model validation process: (A) PCR amplification validation results; (B) RT-PCR technology to verify the relative expression results of Dnah9; (C) The morphology of nasal mucosal cilia of wild-type mice and Dnah9 knockout mice under an optical microscope.

[0016] Figure 3 The changes in various cellular proteins in Dnah9 knockout organoids under immunofluorescence staining technology.

[0017] Figure 4 The effect of nicotinamide adenine dinucleotide on PCD: (A) Morphological changes of cilia after treatment; (B) Effect of treatment on the frequency of ciliary beat. DETAILED DESCRIPTION

[0018] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0019] Example 1. Effect of Nicotinamide Adenine Dinucleotide on the Treatment of Primary Cilia Immobilization Syndrome 1. Experimental methods The present invention refers to the method described in the "Mouse models" section on the left column of page 959 of the prior art (Dynein axonemal heavy chain 10 deficiency causes primary ciliary dyskinesia in humans and mice. Frontiers of medicine, 17(5), 957–971.), and constructs an in vitro model of PCD caused by intrinsic dynein arm deficiency - lung organoids of Dnah9 KO mice. Based on the organoids, an in vitro model for the treatment of PCD is provided, and the effect of nicotinamide adenine dinucleotide on the treatment of primary ciliary dyskinesia is studied. Figure 1 Schematic diagram of the research experiment.

[0020] The specific steps are as follows: 1.1 Primary culture and plating process of lung organoids (1) Prepare digestion solution: Prepare two tubes, each containing 1 ml of digestion solution, which includes Protease E (concentration of 0.25 mg / ml), Collagense I (400 units / ml), Y27632 (10 μmol / L) and DNase (10 units / ml) and set aside.

[0021] (2) Tissue collection and mincing: Remove the lung lobes and trachea from the mouse and place them in PBS solution. Use ophthalmic scissors and forceps to mince the lung and tracheal tissues into small pieces with a diameter of about 2 mm.

[0022] (3) Digestion: Transfer the chopped tissue pieces into the previously prepared digestion solution, place them on a shaker at 37°C, and digest at 100 rpm for 60 min.

[0023] (4) Mechanical blowing: Use a pipette to mechanically blow the digested tissue pieces until all tissue pieces can pass through the pipette tip without resistance. When a large number of suspended cells are observed in the digestion solution, digestion can be stopped.

[0024] (5) Termination of digestion and filtration: Add 300 μl of FBS to the digestion solution to terminate digestion. For tracheal tissue, filter through a 40-μm filter and collect the filtrate; for lung lobe tissue, also filter through a 40-μm filter, but discard the filtrate and use PBS to reversely wash the unfiltered portion on the filter and retain it.

[0025] (6) Washing and preparation of cell suspension: Wash the filtered and eluted tissue, remove the supernatant, and retain the precipitate. Determine the amount of remaining liquid based on the number of wells on the plate and mix it evenly with the precipitate to make a cell suspension.

[0026] (7) Mix Matrigel and cell suspension: Mix the liquefied Matrigel and cell suspension evenly. Depending on the type of well plate (24-well plate, 48-well plate or 96-well plate), add an appropriate amount of Matrigel (30 μl for 24-well plate, 15 μl for 48-well plate, 6 μl for 96-well plate, and the concentration must be greater than 80%). When spreading the plate, pay attention to mixing the tissue and Matrigel so that they are evenly distributed in the Matrigel. After spreading the plate, place the well plate in a 37°C incubator and allow the Matrigel to solidify for 15 minutes.

[0027] (8) Add culture medium: After Matrigel solidifies, add an appropriate amount of culture medium to each well (500 μL for 24-well plate, 200 μL for 48-well plate, 100 μL for 96-well plate). During the culture process, replace the culture medium according to cell growth and color change of the culture medium (when it turns yellow). Generally, replace the culture medium every 4 days.

[0028] 1.2 Organoid passaging steps (1) Collection and centrifugation: Use a pipette to gently scrape the organoids and matrix gel at the bottom of the culture dish and aspirate the resulting suspension into an EP tube. Centrifuge the suspension in the EP tube at 200G for 3 minutes.

[0029] (2) Preliminary treatment: After removing the supernatant, add 1 ml of cell recovery solution to the EP tube and mix it by pipetting. Place the mixed solution on ice for 20 minutes and then centrifuge it again.

[0030] (3) Washing step: After centrifugation, remove the supernatant, add 1 ml of WASH solution, and mix by pipetting. Centrifuge again at 200G for 3 minutes.

[0031] (4) Digestion: After removing the supernatant, add 1 ml of Tryple solution to the EP tube and mix by blowing. Place the EP tube in a 37-degree Celsius incubator for digestion for 5-10 minutes. Take it out every 5 minutes, blow and observe the digestion status. If the digestion is incomplete, the digestion time can be appropriately extended until no white precipitate appears. After digestion is completed, add an appropriate amount of WASH solution to terminate the digestion, and centrifuge again at 200G for 3 minutes.

[0032] (5) Wash again: After centrifugation, pour off the supernatant, add 1 ml of WASH solution, and mix by pipetting. Repeat this step twice, centrifuging at 200G for 3 minutes each time.

[0033] (6) Filtration: Use a 100 μm filter for coarse filtration. This step is suitable for organoids that need to be cultured quickly.

[0034] (7) Glue spreading step: After removing the supernatant, add an appropriate amount of matrigel glue (30ul per well) to the EP tube. After mixing by pipetting, spread the matrigel glue in the wells of the 24-well plate (30ul per well). Place the 24-well plate with glue in a 37°C incubator and let it stand for 15 minutes.

[0035] (8) Add culture medium: Take out the 24-well plate from the incubator and add an appropriate amount of culture medium to each well (0.5 ml per well). Place the 24-well plate with culture medium back into the 37°C incubator for incubation.

[0036] 1.3 Organoids as a platform for drug screening After the lung tracheal organoids were digested into single cells, they were re-spread in a 48-well plate. Subsequently, the drug NAD+ was diluted in the culture medium according to the gradient concentration, and these drug-containing culture media were added to each well. As a control, another group of wells were added with only PBS solution. Each group of experiments was repeated 4 times to ensure the reliability of the data. Drug treatment lasted for one week.

[0037] 1.4 Assessment of ciliary beating Organoids from wild-type mice, Dnah9 knockout (KO) mice, and drug-treated organoids were placed under an upright optical microscope (Olympus CKX41) for observation. The microscope was equipped with a high-speed camera (Sprinter, manufactured by Optronis GmbH) and filmed using a 100x oil-immersion objective. During the filming process, 10 seconds of video were continuously shot at a high speed of 200 frames per second, and then the shooting speed was slowed down to 0.1 times for more detailed analysis.

[0038] 2. Experimental results (1) PCD model verification process The present invention has successfully constructed an organoid system containing a Dnah9 gene knockout. In order to effectively verify the genotype of this system, PCR amplification technology was used, followed by agarose gel electrophoresis for detection. The results showed that the present invention has clearly obtained a Dnah9 homozygous knockout organoid (see Figure 2 A). Further, RT-PCR was used to confirm that the expression of the Dnah9 gene in the organoids had been completely knocked out (see Figure 2 B). Under an optical microscope, the present invention compared the morphology of nasal mucosal cilia of wild-type mice and Dnah9 knockout mice and found that the length of nasal mucosal cilia of Dnah9 knockout mice was significantly shorter and more curly (see Figure 2 C).

[0039] (2) Construction of a Dnah9-knockout lung tracheal organoid system The present invention successfully created an organoid model with Dnah9 gene deletion, and used immunofluorescence labeling technology to perform targeted staining on ciliated cells. In this type of organoid model with downregulated Dnah9 expression, the present invention observed a reduction in the number of cilia, a shortening of their length, and morphological curling changes. In the clinical characteristics of patients with PCD (primary ciliary dyskinesia), it was observed that the level of mucin Muc5ac secreted by goblet cells of the respiratory mucosa was significantly increased. This phenotypic feature was also found in the Dnah9 knockout organoids of the present invention; however, the number and morphology of basal progenitor cells (P63 as a marker protein), secretory Clara cells (CC-10 as a marker protein), and respiratory epithelial cells (KRT5 as a marker protein) did not change (see for details). Figure 3 ).

[0040] (3) Studies evaluating the efficacy of nicotinamide adenine dinucleotide on PCD The present invention uses the Dnah9 gene knockout organoid system as a drug screening platform, selects diphenyliodonium chloride as a negative control, and sets up ATP as a positive control (previous studies have confirmed that ATP can effectively enhance the cilia beating rate at a concentration of 10µM). On this basis, the present invention designs three different concentrations of nicotinamide adenine dinucleotide experimental groups (concentrations are 10µM, 50µM, and 100µM, respectively), and introduces these concentrations of drugs into the Dnah9 knockout organoids. To ensure the robustness of the experimental data, 4 repeated experimental units were set for each concentration level. After 16 consecutive days of drug treatment, the present invention quantitatively evaluated the beating activity of cilia. Figure 4 A intuitively shows the morphological changes of organoids after drug treatment. Figure 4 B revealed that the addition of diphenyliodonium chloride caused the cilia to completely lose their ability to beat, while compared with Dnah9 knockout organoids that did not receive drug treatment, the addition of both nicotinamide adenine dinucleotide and ATP showed a positive effect on the recovery of cilia beating function. After in-depth analysis of the data, it was found that 50µM and 100µM nicotinamide adenine dinucleotide exceeded 10µM ATP in promoting the recovery of cilia beating function, especially 100µM nicotinamide adenine dinucleotide, which induced a significant increase in the frequency of cilia beating, almost approaching the level of cilia beating in normal wild-type mice (see Figure 4 B).

[0041] The above results show that nicotinamide adenine dinucleotide has a significant effect on restoring ciliary motility and can be used to effectively treat primary ciliary immobility syndrome.

[0042] In summary, the present invention discovered for the first time that nicotinamide adenine dinucleotide can effectively restore ciliary motility and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile ciliary syndrome.

Claims

1. Use of nicotinamide adenine dinucleotide in the preparation of a medicament for preventing and / or treating primary immotile ciliary syndrome.

2. The use according to claim 1, characterized in that The drug is a drug that restores the ciliary movement function.

3. The use according to claim 1, characterized in that The drug is a pharmaceutical preparation prepared by taking nicotinamide adenine dinucleotide as an active ingredient and adding pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that The pharmaceutical preparation is an oral preparation or an injection preparation.

5. The use according to claim 4, characterized in that The oral preparations are tablets, granules and capsules.

6. The use according to claim 4, characterized in that The injection preparation is a powder injection.

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