WO2019154220A1 - Use of β-nicotinamide mononucleotide or precursor thereof in preparation of medicament or health care product for treating or alleviating respiratory disorder or disease - Google Patents

Abstract

The present invention belongs to the field of medical technology, and particularly relates to use of β-nicotinamide mononucleotide or a precursor thereof in preparation of a medicament or health care product for treating or alleviating respiratory disorder or disease. The technical problem to be solved by the present invention is to provide a new alternative solution for preparing a medicament or health care product for treating or alleviating respiratory disorder or disease, and the technical solution for solving the technical problem above is to provide use of β-nicotinamide mononucleotide or a precursor thereof in preparation of a medicament or health care product for treating or alleviating respiratory disorder or disease. β-nicotinamide mononucleotide or β-nicotinamide ribose can effectively treat pulmonary diseases such as chronic obstructive pulmonary disease, and can obviously alleviate pulmonary macrophagy ageing caused by inhalable particles, having a good application prospect.

Description

Use of a β-nicotinamide mononucleotide or a precursor thereof for the preparation of a medicament or a health care product for treating or alleviating a respiratory disorder or disease Technical field

The invention belongs to the technical field of medicine, and particularly relates to the use of β-nicotinamide mononucleotide or a precursor thereof for preparing a medicament or a health care product for treating or alleviating respiratory disorders or diseases.

Background technique

Nicotinamide nucleotides are also called β-Nicotinamide Mononucleotide (β-NMN or NMN) or nicotinamide mononucleotide or nicotinamide nucleotides. The molecular formula is as follows:

--nicotinamide mononucleotide (NMN)

Nicotinamide nucleotides are important products of nicotinamide phosphoribosyltransferase (NAMPT) regulation reaction, and key intermediates in NAD+ synthesis process play an important role in cell energy metabolism. And Nicotinamide Riboside (NR) is a precursor of β-nicotinamide mononucleotide (NMN).

--nicotinamide ribose (NR)

It is well known that nicotinamide adenine dinucleotide (NAD+) is an important coenzyme for cell energy conversion and plays an important role in cellular energy metabolism. Nicotinamide riboside (NR) and β-Nicotinamide Mononucleotide (NMN) are key precursors for the synthesis of NAD+ and are important products of nicotinamide phosphoribosyltransferase (NAMPT) regulation. It is naturally found in human cells and in a variety of foods, such as broccoli, curls, and milk, but it is very small. A number of studies have shown that NAD+ has an important relationship with some age-related diseases, aging, and reproductive pregnancy. However, in the case of NAD+, it is difficult to directly feed the human body, and NR and NMN, which are precursors, are easy to synthesize NAD+. Studies have shown that supplementation with NAD+ precursor NMN or NR can increase NAD+ levels in tissues and delay physiological function decline: including inhibition of weight gain, enhancement of energy metabolism, improvement of sensitivity to insulin, improvement of vision, etc., NMN was found to improve aging Causes impaired glucose tolerance in type 2 diabetes; prevents glaucoma in the elderly; NR increases the number of muscle stem cells and exercise capacity.

Chronic obstructive pulmonary disease is a chronic progressive disease caused by chronic inflammation and destruction of the airways and lung parenchyma, and is usually associated with smoking or prolonged exposure to other harmful microparticles and gases. Chronic obstructive pulmonary disease is blocked by airways and comes from The reduction in maximum expiratory flow of the lungs is a major feature. The disease is characterized by a progressive airflow block that is sometimes partially reversed by administration of a bronchodilator. Typical symptoms are coughing, excessive spasm, and difficulty breathing. The term chronic obstructive pulmonary disease encompasses a series of pulmonary activity processes and is a chronic bronchitis and/or emphysema characterized by airflow obstruction.

Worldwide, chronic obstructive pulmonary disease, whether morbidity or mortality, is still increasing year by year, and it is expected to become the world's third leading cause of death by 2030. Among the residents over 40 years old in China, the incidence of chronic obstructive pulmonary disease is as high as 8.2%, ranking second in China due to the cause of death. Therefore, the number of deaths caused by illness has exceeded 1 million in the following year, and this number is rising. the trend of. After the age of 40, the incidence of chronic obstructive pulmonary disease will begin to increase exponentially, and the large number of patients will result in a heavy health care burden.

However, the treatment options and curative effects of chronic obstructive pulmonary disease are very limited, and more frequent symptomatic treatment, often using bronchodilators. There are no measures to fundamentally alleviate the progression of chronic obstructive pulmonary disease. Therefore, there is an urgent need to develop new drugs for the treatment of chronic obstructive pulmonary disease. The current therapeutic drugs for chronic obstructive pulmonary disease are mainly used for symptomatic treatment, and there are no drugs or treatments that can change the underlying inflammation or change the progression of the disease.

Particulate matter refers to a general term for solid and liquid particulate matter floating in the air, and has a particle size ranging from about 0.1 to 100 micrometers. Inhalable particulate matter can be inhaled by the human body and deposited in the respiratory tract, alveoli, etc. to cause disease. Particles with a particle size below 10 microns are often referred to as respirable particles, also known as PM10. The smaller the diameter of the particles, the deeper the area into the respiratory tract. Particles of 10 micron diameter are usually deposited in the upper respiratory tract, 5 micron in diameter can enter the deep part of the respiratory tract, and below 2 microns can penetrate 100% into the bronchioles and alveoli. Common chemical constituents in respirable particulates are particulate elemental carbon (PEC, sometimes referred to as carbon black), inorganic ions, trace elements, and organic compounds, and sometimes inhalable particulate matter also adsorbs pathogenic microorganisms (viruses and bacteria). Inhalable particulate matter enters the body primarily through the respiratory tract, and a small portion can enter the body through the digestive tract or skin. After the inhalable particles are deposited in the human respiratory tract, their clearance, retention and transfer are related to their particle size and deposition location. In general, the smaller the particle size and the further the deposition site, the longer the removal time required, and the easier it is to stay in the human body, the easier it is to transfer toxic substances to other parts of the body.

Carbon black (CB) is one of the main air pollutants in China. It is harmful to humans after inhalation. Combustion is the main source of this particulate matter. Due to huge energy consumption, eastern and northern China is the most serious in the world. One of the aerosol contaminated sites. Carbon black is also an important and typical component of respirable particulate matter in many areas. After CB enters the respiratory tract, most of the particles are cleared by mucociliary movement. However, ultrafine particles are able to penetrate the blood gas barrier and shift to the lungs and systemic circulation. In vivo and in vitro studies have shown that CB can reduce the activity of lung epithelial cells, leading to changes in chronic obstructive pulmonary disease such as chronic bronchitis and emphysema in the lungs. In addition, in the cells and non-cells, carbon black nanoparticles (CBNPs) can induce the production of reactive oxygen species (ROS).

At present, there is no β-Nicotinamide Mononucleotide (NMN) and β-nicotinamide Riboside (NR) for the treatment or alleviation of chronic obstructive pulmonary disease and treatment or alleviation caused by inhalable particles. Related reports of lung diseases, especially lung macrophage aging.

Summary of the invention

The technical problem to be solved by the present invention is to provide a new alternative for preparing drugs or health care products for treating or alleviating respiratory disorders or diseases.

The technical solution of the present invention to solve the above technical problems is to provide a use of a β-nicotinamide mononucleotide or a precursor thereof for the preparation of a medicament or a health care product for treating or alleviating a respiratory disorder or disease.

Wherein, in the above use, the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose.

In the above use, the respiratory disorder or disease is a pulmonary disease.

Among the above uses, the lung disease is a chronic obstructive pulmonary disease.

Among the above uses, the lung disease is a lung disease caused by inhalable particles in the air.

Among the above uses, the lung disease caused by the inhalable particles in the air is lung damage caused by inhalable particles in the air.

Wherein, in the above use, the lung injury is aging of pulmonary macrophages.

Further, in the above application, the air inhalable particles are at least one of PM10 particles, PM2.5 particles or carbon black particles.

In the above application, the drug is prepared by adding a pharmaceutically acceptable excipient or an auxiliary component to the β-nicotinamide mononucleotide or a precursor thereof as an active ingredient.

Further, the preparation is an oral preparation.

Further, the oral preparation per unit contains β-nicotinamide mononucleotide or a precursor thereof of 25-1000 mg.

Further, the oral preparation includes a solid preparation, a liquid preparation or a suspension preparation.

Further, the solid preparation includes a capsule, a tablet, a pill, a powder or a granule.

Further, the liquid preparation includes an emulsion, a solution, a suspension, a syrup or an elixir.

The invention also provides a method of treating or ameliorating a respiratory disorder or disease comprising the step of administering to a subject having a respiratory disorder or disease an effective amount of a beta-nicotinamide mononucleotide or a precursor thereof.

Wherein, in the above method, the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose.

In the above method, the respiratory disorder or disease is a pulmonary disease.

In the above method, the lung disease is chronic obstructive pulmonary disease.

In the above method, the lung disease is a lung disease caused by inhalable particles in the air.

In the above method, the lung disease caused by the inhalable particles in the air is lung damage caused by inhalable particles in the air.

Wherein, in the above method, the lung injury caused by the inhalable particles in the air is aging of the pulmonary macrophage.

Wherein, in the above method, the inhalable particles are at least one of PM10 particles, PM2.5 particles or carbon black particles.

Wherein, in the above method, the administration route in the method is oral administration.

In the above method, the effective amount of the β-nicotinamide mononucleotide or a precursor thereof is 50 to 1000 mg/d.

Wherein, in the above method, the oral administration dosage form comprises a solid preparation, a liquid preparation or a suspension preparation.

Further, the solid preparation includes a capsule, a tablet, a pill, a powder or a granule.

Further, the liquid preparation includes an emulsion, a solution, a suspension, a syrup or an elixir.

The beneficial effects of the invention are:

The present invention creatively provides the use of a beta-nicotinamide mononucleotide and its precursor beta-nicotinamide ribose in the manufacture of a medicament or nutraceutical for treating or ameliorating a respiratory disorder or disease. The present invention found that β-nicotinamide mononucleotide and its precursor can effectively treat or alleviate respiratory disorders or diseases, such as chronic obstructive pulmonary disease, and reduce the occurrence of PM10 particles, PM2.5 particles or carbon black particles. The aging of lung macrophages, which provides a clinical drug for the relief and recovery of lung injury caused by chronic obstructive pulmonary disease as a representative of lung diseases and respirable particles represented by nanocarbon particles. New effective options.

DRAWINGS

Figure 1 shows the results of PPE-induced chronic obstructive pulmonary disease in mice.

Figures 2A and 2B show the therapeutic effects of PPE on chronic obstructive pulmonary disease in mice.

Figure 3 shows the results of mouse lung weight.

Figure 4 shows the results of lung tissue staining.

Figure 5 shows the effect of NMN on lung neutrophils in mice with chronic obstructive pulmonary disease.

Figure 6 shows the effect of NMN on the proportion of lung mononuclear cells in mice with chronic obstructive pulmonary disease.

Figure 7 shows a lung tissue-specific lipase staining pattern.

Figure 8 shows the levels of p16 and p21 proteins in lung lavage cells and lung tissues of PPE-induced chronic obstructive pulmonary disease model mice.

Figure 9 shows the effect of NMN on the expression of p16 and p21 protein in mice with chronic obstructive pulmonary disease; a is lung lavage cell protein, and b is lung tissue protein.

Figure 10 is a diagram showing the immunohistochemical staining of lung tissue in a mouse model of chronic obstructive pulmonary disease.

Figure 11 shows qPCR results in lung tissue of mice with chronic obstructive pulmonary disease.

Figure 12 shows the lung function of mice with chronic obstructive pulmonary disease model. Figure A shows the statistical graph of quasi-static lung compliance (Cchord). In Figure B, Cfvc50 is the lung compliance when 50% of the vital capacity is reached, and CP0 is the pressure. Lung compliance at 0 o'clock.

Figure 13 shows the lung FEV100 values of mice in the chronic obstructive pulmonary disease model group.

Figures 14A-14E show the results of lung MMEF, PEF, IC, ERV, FVC values in mice with chronic obstructive pulmonary disease model group.

Figures 15A and 15B show the expression of γh2ax in alveolar macrophages, which is shown as the percentage (%) of stained cells (**p<0.01).

的表达，图示为染色细胞占的百分比(％)。(**p<0.01)。 Figure 16A, 16B shows the lung cells

The expression is shown as the percentage (%) of stained cells. (**p<0.01).

17A and 17B show changes in macrophage morphogenesis caused by CBNPs (Fig. is cell diameter um, *p<0.05, **p<0.01 compared with control group).

Figure 18 shows the changes in intracellular aging-related protein levels of macrophages treated with CBNPs.

Figure 19 shows the changes in intracellular ROS levels of macrophages treated with different concentrations of CBNPs. Treatment of peritoneal macrophages for 1 week) (****p<0.0001).

Detailed ways

The present invention provides a use of a β-nicotinamide mononucleotide or a precursor thereof for the preparation of a medicament or a health care product for treating or ameliorating a respiratory disorder or disease.

Wherein, in the above use, the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose.

In the above application, the respiratory disorder or disease is a pulmonary disease; further, the pulmonary disease is a chronic obstructive pulmonary disease.

Among them, the lung disease is a lung disease caused by inhalable particles in the air. Further, the lung disease caused by the inhalable particles in the air is lung damage caused by inhalable particles in the air.

Wherein, in the above use, the lung injury is aging of pulmonary macrophages.

Further, in the above application, the air inhalable particles are at least one of PM10 particles, PM2.5 particles or carbon black particles.

The PM10 particles refer to particulate matter having a particle size below 10 microns in air. The PM2.5 particles refer to particulate matter having a particle size below 2.5 microns in air.

In the above application, the drug is prepared by adding a pharmaceutically acceptable excipient or an auxiliary component to the β-nicotinamide mononucleotide or a precursor thereof as an active ingredient.

Further, the preparation is an oral preparation.

Further, the oral preparation per unit contains β-nicotinamide mononucleotide or a precursor thereof of 25-1000 mg.

Further, the oral preparation includes a solid preparation, a liquid preparation or a suspension preparation.

Further, the solid preparation includes a capsule, a tablet, a pill, a powder or a granule.

Further, the liquid preparation includes an emulsion, a solution, a suspension, a syrup or an elixir.

The invention also provides a method of treating or ameliorating a respiratory disorder or disease comprising the step of administering to a subject having a respiratory disorder or disease an effective amount of a beta-nicotinamide mononucleotide or a precursor thereof.

Wherein, in the above method, the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose.

In the above method, the respiratory disorder or disease is a pulmonary disease.

In the above method, the lung disease is chronic obstructive pulmonary disease.

In the above method, the lung disease is a lung disease caused by inhalable particles in the air.

In the above method, the lung disease caused by the inhalable particles in the air is lung damage caused by inhalable particles in the air.

Wherein, in the above method, the lung injury caused by the inhalable particles in the air is aging of the pulmonary macrophage.

Wherein, in the above method, the inhalable particles are at least one of PM10 particles, PM2.5 particles or carbon black particles.

Wherein, in the above method, the administration route in the method is oral administration.

In the above method, the effective amount of the β-nicotinamide mononucleotide or a precursor thereof is 50 to 1000 mg/d.

Wherein, in the above method, the oral administration dosage form comprises a solid preparation, a liquid preparation or a suspension preparation.

Further, the solid preparation includes a capsule, a tablet, a pill, a powder or a granule.

Further, the liquid preparation includes an emulsion, a solution, a suspension, a syrup or an elixir.

The above "subject" in the present invention means any organism to which a drug according to the present invention can be administered, for example, for the purpose of experimentation, diagnosis, prevention, and/or treatment. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Subjects may seek treatment or necessary treatment, need treatment, receive treatment, receive treatment in the future, or be treated by a trained professional for a particular disease or condition.

In the present invention, "treating" means therapeutic treatment and prophylactic or preventative measures, the object of which is to prevent or delay (reduce) an unwanted physiological condition, disorder or disease, or to obtain a beneficial or desired clinical condition. result. Clinical outcomes that are beneficial or desirable include, but are not limited to, relief of symptoms; amelioration of the extent of the condition, disorder, or disease; stability of the condition, disorder, or state of the disease (ie, no deterioration); condition, disorder, or Delay or slowing of the onset of disease progression; improvement or relief of a condition, disorder, or disease state (whether partial or total), whether detectable or undetectable; improvement of at least one measurable human physiological parameter The parameter need not be discernible by the patient; or an illness, an improvement, or an improvement in the condition, disorder, or disease. Treatment involves causing a clinically significant response without excessive levels of side effects.

The β-nicotinamide mononucleotide of the present invention or a precursor thereof, such as β-nicotinamide ribose, is not particularly limited in the treatment or relief of pulmonary diseases, and representative administration methods include, but are not limited to, oral administration, intravenous administration. Parenteral administration and topical administration, intramuscular or subcutaneous.

Solid preparations for the preparation of oral preparations include capsules, tablets, pills, powders or granules. In these solid preparations, the β-nicotinamide mononucleotide or a precursor thereof is used as an active ingredient, and is mixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or the following Ingredient mixing: (a) filler or compatibilizer, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binder, for example, hydroxymethylcellulose, alginate, gelatin, polyethylene (i) a humectant, for example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and carbonic acid (e) a slow solvent such as paraffin; (f) an absorption accelerator such as a quaternary amine compound; (g) a wetting agent such as cetyl alcohol and glyceryl monostearate; (h) an adsorbent, for example, Kaolin; and (i) a lubricant such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

Solid preparations such as tablets, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner.

The liquid preparations include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances. In addition to these inert diluents, liquid dosage forms may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfumes.

The suspending agent of the present invention may further comprise ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, microcrystalline cellulose, aluminum methoxide or agar.

In addition to the above dosage forms, compositions for parenteral injection can also be prepared. A physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion may be employed. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

The pharmaceutically acceptable excipient or auxiliary component of the present invention means a substance which is contained in a dosage form other than the active ingredient. It may refer to at least one of a carrier, carrier, diluent or adjuvant species that is generally chemically or physically compatible with the other components that make up a pharmaceutical dosage form and that is physiologically compatible with the receptor.

The pharmaceutically acceptable auxiliary component of the present invention has certain physiological activity, but the addition of the component does not change the dominant position of the above-mentioned pharmaceutical composition in the course of disease treatment, but only plays an auxiliary effect, and these auxiliary effects are only The use of known activity of this component is an adjuvant treatment that is commonly used in the medical field. It is still within the scope of the present invention to use the above auxiliary ingredients in combination with the pharmaceutical composition of the present invention.

The specific embodiments of the present invention are further explained by the following examples, but the scope of the present invention is not limited to the scope of the embodiments.

The reagents used in the examples were all commercially available products unless otherwise stated.

Example 1 Therapeutic effect of NMN and NR on chronic obstructive pulmonary disease

(1) Establishment of a mouse model of chronic obstructive pulmonary disease

Animals: 6-8 weeks, C57 mice;

Reagent: porcine pancreatic elastase solution PPE (USA, Sigma), diluted in sterile PBS;

method:

1) Anesthetize the mouse (add isoflurane at a rate between 1.5-2 L/min), remove it from the box and place it in the supine position, carefully open its mouth and rubber band attached to the upper incisor , lift the rubber band from the nail.

2) Using a small forceps, gently grasp the mouse's tongue, pull it out of the mouth 0.5 to 1 cm, and drop the elastase solution (50 μL) on the distal end of the oropharynx, behind the tongue.

3) Hold the tongue with one hand and block the two nostrils with the other hand for 5-6 seconds. With the extension of the tongue and the blockage of the nose, the mice will not be able to swallow and the elastase solution will be inhaled. Lower respiratory tract.

4) Release the mouse tongue, gently massage the mouse chest, the elastase solution flows down and distributes throughout the intestinal airway, removes the rubber band, and puts the mouse back.

5) Observe for 5 to 10 minutes, pay attention to the recovery status and respiratory status of the mice.

C57 mice were induced with chronic obstructive pulmonary disease with different doses of PPE (0.68 U, 1.00 U, 1.50 U). The result is shown in Figure 1. It can be seen from the figure that after the PBS tongue instillation, there is no difference between the mouse lung and the normal mouse lung, and after the PPE tongue instillation, the lung of the mouse is significantly larger than the PBS group, and it is quantitatively dependent, when the PPE measurement reaches 1.5. At U, there was significant necrosis in the lungs of mice.

(two) experimental grouping and administration

Grouping:

The experiment was divided into the control group (PBS group, PBS tongue instillation), model group (PPE group, PPE post-tongue instillation to establish a chronic obstructive pulmonary disease model), treatment group (PPE + NMN group, treated with NMN chronic Obstructive pulmonary disease model mice), n=5.

Dosing:

--nicotinamide mononucleotide (NMN, USA, Sigma), dissolved in PBS, 0.10 g/mL, gavage, 200 μL/only (20 mg/day*day). The treatment group was given the above oral NMN drug, the control group and the model group were given an equal volume of PBS. The administration time was 30 days.

(3) Test of experimental results

1. Measurement of lung mechanics

Determine the mechanical properties of the lungs:

Mice were weighed and deeply anesthetized by intraperitoneal injection of pentobarbital (90 mg/kg) and pancuronium bromide (0.5 mg/kg). The trachea was dissected, the trachea was inserted into the cannula, and the trachea was tied to the cannula with a suture. The cannula was attached to a computer-controlled small animal ventilator, and the lung function related parameters of the mouse were determined, and each parameter was measured three times. After the assay was completed, the mice were removed and the lungs were removed for paraffin sectioning, protein extraction and qPCR.

Mice in which PPE induces chronic obstructive pulmonary disease are treated with β-nicotinamide mononucleotide (NMN). The results are shown in Figures 2A and 2B. 2A and 2B, the lungs of the chronic obstructive pulmonary disease model group (PPE group) were significantly increased compared with the PBS group, and the PPE-induced chronic obstructive pulmonary disease model was successfully established. Chronic obstructive pulmonary disease was induced in PPE, and it was shown that oral administration of 20 mg/day ORAL (PPE + NMN 20 mg) in mice was effective, and the lungs of the mice were significantly improved compared with the chronic obstructive pulmonary disease model group.

The lung weight statistics are shown in Figure 3. Figure 3 shows that the PPE group of the chronic obstructive pulmonary disease model was significantly increased compared with the PBS control group, and the oral administration of NMN at the same time as PPE administration significantly reduced the lung weight of the mice. *P<0.05, **P<0.01.

2, lung lavage (BAL)

Mice were sacrificed after anesthesia, the trachea was dissected in the neck, and the lungs were lavaged 3 times with 0.6 mL of 0.9% sodium chloride. The lavage fluid was combined, the lavage fluid was centrifuged, 1200 rpm, 5 min, and the supernatant was collected, dispensed, and stored frozen at -80 ° C for subsequent analysis. The lung lavage (BAL) pellet was resuspended in 1 mL of 0.9% sodium chloride and the total number of cells was determined by counting on a hemocytometer. The cells were washed once with 0.9% sodium chloride and the cells were used for protein extraction.

3. Detection of neutrophils in lung tissue and BLA

Single cell preparation of lung tissue: The whole lung tissue of the mice was taken out, the blood was washed away in 0.9% sodium chloride, the excess tissue was removed, the lungs were cut, and 5 mL of 1 mg/mL type I collagenase was digested at 37 ° C for 1 hour. It was filtered through a 70 μm sieve, centrifuged at 1200 rpm for 5 min, and 3 mL of red cracking solution was added for 3 minutes, and washed twice with PBS. The cells were counted by a hemocytometer, and macrophages, monocytes, T cells, and neutrophil flow detection antibodies were added for staining at 4 ° C for 30 min. After washing twice with PBS, the machine was tested.

Neutrophils were stained in lung tissue and BLA, washed twice and then tested on the machine.

Lung tissue staining is shown in Figure 4. HE staining showed that the bronchial and alveolar structures in the control group were clear, the mucosal epithelial cells were arranged neatly, and the alveolar size was uniform. The bronchial epithelial cells in the chronic obstructive pulmonary disease model group showed shedding, the alveolar wall collapsed, and the alveolar cavity showed irregular expansion. Different degrees of inflammatory cell infiltration occur around the airway wall and in the interstitial lung. The bronchial mucosa shrinks, protrudes into the lumen, the lumen narrows or occludes, and mucus is visible in the lumen. Pathological changes in the lungs of mice were significantly alleviated after NMN.

The lung tissue of the mice was cut and stained with 1 mg/mL type I collagenase and stained for flow detection (Fig. 5 and Fig. 6). PPE induced neutrality in lung tissue of mice with chronic obstructive pulmonary disease. The proportion of granulocytes was 18.74%, which was significantly higher than that of the control group (5.04%). After oral administration of NMN, the proportion of neutrophils decreased significantly. *P<0.05, **P<0.01, ***P<0.001.

4, lung morphology (Lung morphometry)

The lungs of the mice were removed and fixed with 4% PFA (tetrafluoroethylene-perfluoroalkoxy vinyl ether copolymer). The fixed lungs were dehydrated, embedded in paraffin, and cut into 3 μm paraffin sections using a rotary microtome. Lung sections were H&E stained and analyzed using Meta Morph software.

Paraffin sections of mouse lung tissue were subjected to specific lipase staining, and the results are shown in Fig. 7. The results showed that the number of neutrophils in the lung tissue of the PPE group increased compared with the PBS control group, and the number of neutrophils decreased significantly after NMN administration.

5, preparation of the whole cell lysate (Preparation of hole cell lysate protein)

Whole cell lysates and nuclear proteins were prepared from lung tissue and BAL cells.

Preparation of lung tissue protein: 0.1 g of mouse lung tissue was taken, and 0.5 ml of radioimmunoprecipitation assay (RIPA) buffer (50 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.25% deoxycholate) was added. , 1 mmol/l Na ₃ VO ₄ , 1 mmol/l NaF, 1 mg/l leupeptin, 1 mg/l aprotinin and 1 mmol/l PMSF), homogenized with tissue homogenizer, kept on ice for 45 min In order to completely lyse the cells. After the lysis was completed, the cells were centrifuged at 13,000 rpm for 15 minutes, and the supernatant was collected as a tissue protein extract for use. The total protein content was determined by BCA (n-butyl polycyanoacrylate) method, and the protein concentration of all samples was adjusted with reference to the lowest concentration.

BAL cell protein preparation: 50 μL of RIPA buffer was added to BAL cells, lysed on ice for 30 min, and then vortexed for 15 seconds. After centrifugation at 13,000 rpm for 5 min, the supernatant was collected as a whole cell lysate for use.

6, immunoblotting (Immunoblot.)

1) Protein samples from lung tissue homogenate and BAL cell lysate were separated by 12.5% SDS-PAGE gel.

2) The isolated protein was electroblotted onto a 0.2 μm nitrocellulose membrane.

3) The membrane was blocked with 5% skim milk for 1 hour at room temperature, washed 3 times with TBST, anti-p21, anti-p16 antibody was added, and incubated overnight at 4 °C.

4) After washing 3 times (5 min each time), incubate with a horseradish peroxidase-labeled secondary antibody (diluted with 5% skim milk, 0.1% Tween 20 [v/v] in PBS 1: 10,000), incubate at room temperature 1h.

5) After washing 3 times (10 min each time), the developing solution was wetted and exposed on an exposure machine.

The sample was loaded equally by quantifying the protein and β-actin hybridization.

The results are shown in Figures 8 and 9. As can be seen from the figure, the levels of p16 and p21 proteins in lung lavage cells and lung tissues of PPE-induced chronic obstructive pulmonary disease model mice were significantly increased, and the levels of p16 and p21 proteins were decreased after oral administration of NMN. *P<0.05, **P<0.01, ***P<0.001, versus PBS;###P<0.001,versus PPE.

7. Immunohistochemical staining of frozen sections

1) Frozen sections (4 μm) were fixed in methanol precooled at 4 ° C for 10 min.

2) Wash PBS (pH 7.2 to 7.4) 3 times for 5 minutes each time.

3) Soak the sections in 3% hydrogen peroxide solution, protected from light for 15 min.

4) Incubate the sections with 5% normal goat serum for 30 minutes to block non-specific binding of the antibody to the tissue sections.

5) Discard the serum and incubate the lung tissue sections with a 1:100 dilution of p21 or a 1:50 dilution of P16 antibody at 4 °C overnight;

6) After washing with PBS, sections were incubated with biotinylated anti-rabbit Ig secondary antibody for 1 hour.

7) DAB staining solution develops color, and washes the slice with tap water to stop color development.

8) Perform hematoxylin counterstaining and microscopic examination.

Immunohistochemical staining (shown in Figure 10) of frozen sections (4-6 μm, original magnification *100) revealed that p16 and p21 were expressed in lung tissue of mice with chronic obstructive pulmonary disease model, after oral administration of NMN, p16 Both the levels of p21 and protein were decreased.

qPCR was found (shown in Figure 11): p16 protein was expressed in the lung tissue of mice with chronic obstructive pulmonary disease model, and the expression level of p16 protein was significantly decreased after oral administration of NMN. *P<0.05, **P<0.01, versus PBS, #P<0.05, vers PPE.

8. Effects on lung function in mice

In the lung function test of mice (Fig. 12), the lung compliance of the model group of chronic obstructive pulmonary disease was significantly lower than that of the PBS group. After NMN administration, the lung compliance of the mice in the treatment group was significantly increased. *P<0.05, **P<0.01.

The lung FEV100 value of the chronic obstructive pulmonary disease model group was significantly lower than that of the PBS group, and the treatment group was significantly increased after NMN administration. *P<0.05, **P<0.01 (Fig. 13).

In addition, the levels of MMEF, PEF, IC, ERV, and FVC in the lungs of the chronic obstructive pulmonary disease model group were significantly lower than those in the PBS group, and the treatment group was significantly increased after NMN administration. *P<0.05, **P<0.01 (Fig. 14).

The results of Example 1 demonstrate in many ways that NMN is effective in the treatment of chronic obstructive pulmonary disease. It has also been found that nicotinamide riboside (NR) also significantly reduces the pathological changes of chronic obstructive pulmonary disease in mice, and thus treats chronic obstructive pulmonary disease.

Example 2 β-nicotinamide mononucleotide NMN and β-nicotinamide ribose NR reduce the aging of pulmonary macrophages caused by carbon particles and the like

(1) Extraction of C57BL/6 macrophages

C57BL/6 alveolar macrophages were extracted, and the ribs of the mouse thoracic cavity were cut out to facilitate the full expansion of the lungs. Then the trachea was separated, and a small opening was cut along the annular cartilage. The smoothed 20 ml empty needle was inserted into the trachea to absorb 1 ml of physiology. Brine, inject the trachea, hold the needle with needle holder, pump back, repeat 2 times, centrifuge 1000 rpm, treat for 5 min, wash once in DMEM, transfer to culture dish, add DMEM (containing calf serum), wait 2 hours after adherence The original medium is discarded, and the obtained adherent cells are alveolar macrophages. C57BL/6 peritoneal macrophages were extracted, and 10 ml of normal saline was injected into the peritoneal cavity of the mice. The saline and the abdominal cavity were fully contacted on both sides of the abdominal cavity of the mice, and physiological saline was drawn into the BD tube. The liquid was centrifuged, 1,000 rpm, 5 min, resuspended in DMEM medium and washed once, then resuspended in DMEM medium containing 10% calf serum, and incubated for 2 h to remove the culture medium containing the suspended cells. After washing once with DMEM medium, adherent macrophages were obtained.

(2) Test procedures and results

1. Carbon nanoparticle induced macrophage aging and inhibition of NR on its aging

CBNPs induce aging of lung macrophages and verify whether NR inhibits the aging of CBNPs on macrophages, using γh2ax antibody (1:5000 dilution) and Sa-beta-gal antibody (1:50 dilution) for three groups. Immunofluorescence staining, macrophages were treated differently for one week, the original medium was removed, the slides were taken out, washed once with PBS, and pre-cooled with -20 ° C for 15 min. After fixation, wash 3 times with PBS for 5 min each time. Subsequently, PBS diluted γh2ax antibody was added and incubated overnight at 4 ° C in a wet box. Wash PBS 3 times for 5 min each, then add FITC-labeled and CY3-labeled fluorescent secondary antibody (1:1000 dilution), respectively, and incubate in a wet box for 2 h at 4 °C. After DAPI was stained and rinsed in PBS, it was observed under an upright fluorescence microscope and photographed. It was found that the alveolar macrophages of C57 mice (Fig. 15A and 15B) were more incubated with CBNPs for 1 week, and the proportion of cells containing fluorescent cells was higher than that of the untreated control cells and NR groups. The percentage of observations in the high-power microscope showed that the positive cells of the CBNPs group were higher than the control group and the NR group. Therefore, it is indicated that CBNPs can promote the aging of macrophages, while NR can inhibit the aging effects of CBNPs on macrophages.

)溶液加入新鲜提取的C57小鼠的巨噬细胞并在24孔板中孵育1周后，直接加入

作液进行sa-beta-gal染色，37摄氏度无二氧化碳的孵箱中孵育1h左右。结果如图16A、16B所示，通过sa-beta-gal染色液直接于24孔板中染色，并于倒置荧光显微镜下观察，观测到部分细胞质蓝染，C57BL/6小鼠的肺巨噬细胞于高倍镜下观测蓝染的阳性细胞的百分比，发现加入CBNPs悬液的实验组，老化细胞比例大于对照组，经NR处理组老化的细胞减少。 We hypothesized that carbon nanoparticles cause aging of macrophages, and NR has the effect of inhibiting its aging. To further confirm the effect of CBNPs on macrophages, 20lCbnps (final concentration is

Add the freshly extracted C57 mouse macrophages to the solution and incubate in a 24-well plate for 1 week.

The solution was stained with sa-beta-gal and incubated for about 1 h in a 37 ° C incubator without carbon dioxide. The results are shown in Figures 16A and 16B, stained directly in a 24-well plate by sa-beta-gal staining, and observed under an inverted fluorescence microscope, and partial cytoplasmic blue staining was observed, and pulmonary macrophages of C57BL/6 mice were observed. The percentage of blue-stained positive cells was observed under high power microscope. It was found that in the experimental group with CBNPs suspension, the proportion of aged cells was larger than that of the control group, and the number of aged cells in the NR-treated group was decreased.

的cbnps悬液，将

溶液加入新鲜提取的C57小鼠的腹腔巨噬细胞并在24孔板(生长面积2cm ²)中孵育1周后观测其细胞形态变化。进一步发现(如图17A和17B所示)，巨噬细胞形态发生变化，巨噬细胞直径增大，细胞突触增多，细胞活化，但经NR处理组的体积变大的细胞减少。 In order to observe the effect of CBNPs on cell morphology, the concentration was 0.05 mg/ml in DMEM medium and diluted in a Petri dish.

Cbnps suspension, will

The solution was added to peritoneal macrophages of freshly extracted C57 mice and observed for cell morphology in a 24-well plate (growth area 2 cm ² ) for 1 week. It was further found (as shown in Figs. 17A and 17B) that the morphology of macrophages changed, the diameter of macrophages increased, cell synapses increased, and cells were activated, but the cells of the NR-treated group were reduced in size.

To further verify the effects of CBNPs and NR on macrophages, we examined the aging-associated proteins in cells obtained after one week of macrophage treatment, as shown in Figure 18: P-Src in the CBNPs group in Western experiments. (Y418)/P-Src (Y529) and sa-beta-gal were the highest, followed by the NR group and the lowest in the Control group. It was further confirmed that CBNPs can induce macrophage aging. At the same time, NR can inhibit the aging of CBNPs.

2. Carbon nanoparticles cause an increase in macrophage ROS

)±NR(浓度0.5mM)处理细胞20min后。或者以CBNPs(浓度为

)±NR(浓度0.5mM)处理细胞1周，去除原培养基，无血清DMEM洗3遍，并加入H2DCF-DA探针(终浓度为0.1μM)，于37℃避光孵育30min，胰酶消化收集贴壁细胞后上机检测。 Previous studies have shown that CBNPs can induce aging of alveolar macrophages by immunofluorescence, Western and sa-beta-gal staining. Moreover, NR has an aging effect of inhibiting CBNPs. To further investigate the oxidative stress of macrophages treated with CBNPs, the changes of intracellular reactive oxygen species in macrophages treated with CBNPs were determined by flow cytometry. The method is as follows: The intracellular reactive oxygen species concentration is determined by H2DCF-DA. Different concentrations of CBNPs (concentrations are

) The cells were treated with ±NR (concentration 0.5 mM) for 20 min. Or with CBNPs (concentration is

The cells were treated with ±NR (concentration: 0.5 mM) for 1 week, the original medium was removed, and the serum-free DMEM was washed 3 times, and the H2DCF-DA probe (final concentration: 0.1 μM) was added, and the mixture was incubated at 37 ° C for 30 min in the dark. After collecting and collecting the adherent cells, they were tested on the machine.

The experimental results are shown in Fig. 19: the concentration of active oxygen in the cells increased after one week of treatment with CBNPs, and NR inhibited the concentration of active oxygen in the cells. It is shown that CBNPs can stimulate the increase of ROS, and NR can inhibit this effect. The results of Example 2 demonstrate in many ways that nicotinamide riboside (NR) NR can effectively alleviate the aging of lung macrophages caused by nano carbon particles. It has also been found that β-Nicotinamide Mononucleotide (NMN) also significantly reduces the aging of lung macrophages caused by nano carbon particles.

It can be known from the embodiments of the present invention that inhalable particles in the air, such as PM10 particles, PM2.5 particles or carbon black particles, cause aging of pulmonary macrophages, which in turn causes lung damage, leading to chronic obstructive pulmonary disease or respiratory disorders. It has been found that β-nicotinamide mononucleotide or its precursor (β-nicotinamide ribose) has the effect of alleviating the above lung damage, and therefore the present invention provides a β-nicotinamide mononucleotide or a precursor thereof in the preparation of a treatment or The use of drugs or health supplements for relieving respiratory disorders or diseases provides a new therapeutic option for the treatment of lung disease-like lung diseases caused by inhalable particles, which is of great significance.

Claims (27)

Use of a β-nicotinamide mononucleotide or a precursor thereof for the preparation of a medicament or health care product for treating or ameliorating a respiratory disorder or disease. The use according to claim 1, characterized in that the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose. The use according to claim 1 or 2, characterized in that the respiratory disorder or disease is a pulmonary disease. The use according to claim 3, characterized in that the lung disease is a chronic obstructive pulmonary disease. The use according to claim 3 or 4, characterized in that the lung disease is a lung disease caused by inhalable particles. The use according to claim 5, characterized in that the lung disease is lung damage. The use according to claim 6, characterized in that said lung injury is aging of pulmonary macrophages. The use according to any one of claims 5 to 7, wherein the inhalable particles are at least one of PM10 particles, PM2.5 particles or carbon black particles. The use according to any one of claims 1 to 8, wherein the drug is a β-nicotinamide mononucleotide or a precursor thereof, and a pharmaceutically acceptable excipient or an auxiliary component is added. Prepared preparation. The use according to claim 9, characterized in that the preparation is an oral preparation. The use according to claim 10, characterized in that each unit oral preparation contains β-nicotinamide mononucleotide or a precursor thereof of 25-1000 mg. The use according to claim 10 or 11, wherein the oral preparation comprises a solid preparation, a liquid preparation or a suspension preparation. The use according to claim 12, characterized in that the solid preparation comprises a capsule, a tablet, a pill, a powder or a granule. The use according to claim 12, characterized in that the liquid preparation comprises an emulsion, a solution, a suspension, a syrup or an elixir. A method of treating or ameliorating a respiratory disorder or disease, comprising the step of administering to a subject having a respiratory disorder or disease an effective amount of a β-nicotinamide mononucleotide or a precursor thereof. The method according to claim 15, wherein the β-nicotinamide mononucleotide precursor is β-nicotinamide ribose. The method according to claim 15 or 16, wherein the respiratory disorder or disease in the method is a pulmonary disease. The method according to claim 17, wherein the lung disease in the method is chronic obstructive pulmonary disease. The method according to claim 17, wherein the lung disease in the method is a lung disease caused by inhalable particles in the air. The method according to claim 19, wherein the lung disease caused by the inhalable particles in the air is lung damage caused by inhalable particles in the air. The method according to claim 20, wherein the lung damage caused by the inhalable particles in the air in the method is aging of the pulmonary macrophage. The method according to any one of claims 19 to 21, wherein the inhalable particles in the method are at least one of PM10 particles, PM2.5 particles or carbon black particles. The method of claim 15 wherein the route of administration in the method is oral administration. The method according to claim 15, wherein the β-nicotinamide mononucleotide or a precursor thereof is present in an effective amount of from 50 to 1000 mg/d. The method according to claim 23, wherein said oral administration form comprises a solid preparation, a liquid preparation or a suspension preparation. The method according to claim 25, wherein said solid preparation comprises a capsule, a tablet, a pill, a powder or a granule. The method of claim 25 wherein said liquid formulation comprises an emulsion, a solution, a suspension, a syrup or an elixir.

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