Application of nicotinamide adenine dinucleotide in the preparation of drugs for treating keloid

Abstract

The present invention discloses the application of nicotinamide adenine dinucleotide in the preparation of drugs for treating keloids. By studying the pathophysiological mechanism of keloids, it is found that there are metabolic reprogramming changes in the mechano-related fibroblasts of keloid patients. There is metabolic reprogramming caused by mitochondrial dysfunction in the main pathogenic fibroblast population of keloids, especially the enhanced intracellular glycolysis level and the accumulation of lactic acid. The present invention searches for and verifies a drug that can regulate glycolysis and mitochondrial metabolism of keloid fibroblasts through cell and animal experiments - NAD+. NAD+ inhibits the development of keloids by improving mitochondrial dysfunction, reducing lactic acid accumulation, inhibiting fibroblast migration and collagen synthesis. The present invention first applies the common drug NAD+ with good safety to keloids. This scheme has small toxic and side effects and also has positive effects on antioxidation, anti-aging and anti-cancer, providing a new scheme for the drug treatment of keloids.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of nicotinamide adenine dinucleotide in the preparation of drugs for treating keloids. Background Art

[0002] Keloid is a skin fibroproliferative disorder characterized by persistent local inflammation and excessive deposition of collagen. These scar tissues usually present a raised morphology and have obvious invasive growth characteristics, with features such as irregular edges and pigmentation. The growth of keloids not only causes changes in the appearance of the skin but is usually accompanied by itching, pain, and other discomfort. These symptoms often have a negative impact on the mental and emotional state of patients, thus affecting their daily life and social activities. Research shows that the presence of keloids is closely related to the quality of life of patients, and patients often feel inferior and anxious, leading to mental health problems.

[0003] There are various treatment methods for keloids, and currently, surgical resection, hormone injection, and radiotherapy and chemotherapy are still the mainstream. Surgical resection is considered one of the main treatment methods. Although most patients can obtain certain improvement after surgery, the recurrence rate is relatively high, often making the surgical effect questioned. Hormone injection, especially steroid drugs such as prednisone and triamcinolone acetonide, can effectively inhibit the proliferation of keloids, and the operation is relatively simple and safe. However, the hormone treatment course is long, and the manifestation of the effect is often delayed, with a high clinical recurrence rate and many adverse reactions. In addition, radiotherapy and chemotherapy show certain effects in reducing the lesion, but due to their large side effects, they are limited in clinical application, especially in young or patients with impaired liver and kidney function, where they may damage their normal tissues.

[0004] Although there are various current treatment methods for keloids, the preventive measures against its occurrence or recurrence are still insufficient, which makes patients still face a high recurrence risk after treatment. Therefore, it is particularly important to explore preventive drugs for keloids. The research and development of such drugs can not only reduce the incidence of keloids but also delay the further development of existing keloids, thereby effectively improving the quality of life of patients. Through in-depth research on the mechanism of scar formation, the targeted development of new drugs may provide new treatment options for keloid patients and ultimately achieve individualized and comprehensive health management. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of nicotinamide adenine dinucleotide in the preparation of drugs for treating keloids. Nicotinamide adenine dinucleotide can inhibit the glycolysis of keloid fibroblasts, reduce lactic acid production and extracellular matrix synthesis, and inhibit the occurrence and development of keloids.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides an application of nicotinamide adenine dinucleotide in the preparation of a medicament for treating keloid.

[0008] To explore the drug action targets, we carefully explored the pathophysiological mechanism of keloid. To explore effective new drugs, we seriously studied the pathophysiological mechanism of keloid. In the study population, we found that there were metabolic reprogramming changes in the mechanosensitive fibroblasts of keloid patients. Further animal and cell experiments proved that one of the pathogenesis of the occurrence and development of keloid was that under tensile stimulation, mitochondrial dysfunction in keloid fibroblasts induced an increase in LDHA expression, resulting in lactate accumulation, thereby promoting fibroblast proliferation and enhancing myofibroblastization.

[0009] In view of our finding that the enhancement of mechanosensitive mitochondrial dysfunction and glycolytic capacity promotes the occurrence and development of keloid, we screened 404 compounds approved by the US Food and Drug Administration (FDA) related to mitochondria and glycolysis to identify potential therapeutic agents. We cultured primary keloid fibroblasts for high-throughput drug screening. High-content cell imaging technology was used to evaluate the effects of these compounds on the expression of LDHA and COL1A1 in fibroblasts after tensile stimulation. Among the screened drugs, we found that a drug, nicotinamide adenine dinucleotide (NAD + ), could significantly reduce the expression of LDHA and COL1A1. Further cell and animal experiments proved that NAD + inhibited the glycolysis of keloid fibroblasts, reduced lactate production and extracellular matrix synthesis, and inhibited the occurrence and development of keloid (as Figure 1 shown).

[0010] Preferably, the medicament is for topical skin administration.

[0011] Preferably, the dosage form of the medicament includes ointment, gel, patch, emulsion or injection.

[0012] Preferably, the medicament includes nicotinamide adenine dinucleotide and excipients.

[0013] Preferably, the nicotinamide adenine dinucleotide can inhibit the glycolysis of keloid fibroblasts.

[0014] Preferably, the nicotinamide adenine dinucleotide can reduce lactate production and extracellular matrix synthesis.

[0015] NAD +is an important coenzyme for redox reactions, and a decrease in its level is closely related to the occurrence of aging and fibrotic diseases. NAD + plays a central role in maintaining processes such as cell metabolism, energy production, and gene repair. NAD + Currently, it has a wide range of application prospects in clinical practice, covering areas such as aging, kidney diseases, cardiometabolic diseases, neurodegenerative diseases, inflammation, fibrosis, and cancer. In vitro experiments have shown that NAD + has effects such as inhibiting glycolysis, reducing lactic acid production, inhibiting collagen synthesis, and anti-fibrosis.

[0016] Preferably, in the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 50 - 150 mg / kg.

[0017] Preferably, in the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 100 mg / kg.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By studying the pathophysiological mechanism of keloid, it is found that there are metabolic reprogramming changes in the mechano-related fibroblasts of keloid patients. Our research shows that there is metabolic reprogramming caused by mitochondrial dysfunction in the main pathogenic fibroblast population of keloid, especially the enhanced intracellular glycolysis level and the accumulation of lactic acid. The present invention has searched for and verified through cell and animal experiments a drug - NAD + , NAD + which can regulate glycolysis and mitochondrial metabolism of keloid fibroblasts. By improving mitochondrial dysfunction, reducing lactic acid accumulation, inhibiting the migration and collagen synthesis of fibroblasts, and inhibiting the development of keloid. The present invention first applies this common drug with good safety - NAD + to keloid. This scheme has small toxic and side effects, and at the same time has a positive effect on antioxidant, anti-aging, and anti-cancer, providing a new scheme for drug treatment of keloid. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a diagram of the mechanism of action of NAD + ;

[0021] Figure 2 is a diagram of the situation of nude mice in the control group and the drug - administered group on the 21st day after modeling;

[0022] Figure 3 is a diagram of the keloid nodules of nude mice in the control group and the drug - administered group on the 21st day after modeling;

[0023] Figure 4 is a growth curve diagram of the keloid nodules of nude mice in the control group and the drug - administered group;

[0024] Figure 5 Line graph of lactic acid production in primary keloid fibroblasts of the control group and the drug group after mechanical stretching

[0025] Figure 6 Graph of the degree of lactate modification and extracellular matrix synthetic proteins in primary keloid fibroblasts of the control group and the drug group after mechanical stretching Detailed implementation manners

[0026] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments

[0027] It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are all purchased through commercial channels

[0028] Example 1 NAD + In vitro experimental study on reducing the degree of fibrosis of keloid

[0029] (1) Establishment of humanized keloid mouse model and administration

[0030] After obtaining the consent of the patient and signing the informed consent form, local anesthesia was performed using aseptic technique, and then skin lesion tissue samples were obtained from the diagnosed keloid patients. The excised keloid tissue was placed in sterile physiological saline containing antibiotics to prevent bacterial infection, and then centrifuged, washed and cut to prepare tissue blocks of 1 mm in size

[0031] Immunodeficient nude mice were selected and anesthetized with 1% sodium pentobarbital at a dose of 50 mg·kg -1 . After anesthesia, a surgical incision was prepared subcutaneously on the back of the mice, the surgical area was cleaned and disinfected, and the processed keloid tissue blocks were transplanted into the subcutaneous tissue of the mice. The skin incision was sutured using fine needles and sutures to construct a humanized keloid mouse model. One week after modeling, it was observed that keloid-like nodules appeared significantly on the back of the modeled mice, indicating successful modeling. Twelve of the successfully modeled mice were given intralesional injections of 50 mg / kg, 100 mg / kg, and 150 mg / kg NAD + for 21 consecutive days. In addition, 3 mice in the control group were not treated, and a total of 15 mice were given regular feed and water

[0032] (2) Determination of relative scar inhibition rate

[0033] The areas of keloid nodules of each nude mouse were measured at 0, 5, 10, and 15 days after administration. The keloid nodules were recorded using a camera at a fixed height, and a growth curve of the keloid nodules was plotted

[0034] Figure 2This is a diagram showing the situation of nude mice in the control group and the drug administration group on the 21st day after modeling. The addition amount of yoda1 (a specific activator of piezo1, which increases the environmental mechanical responsiveness) is 1 mg / kg once every other day. Figure 3 This is a diagram showing the keloid nodules of nude mice in the control group and the drug administration group on the 21st day after modeling. Figure 4 This is a growth curve diagram of keloid nodules of nude mice in the control group and the drug administration group. From Figure 3 and Figure 4 the results, it can be seen that compared with the control group, the nodule growth rate, volume and mass of mice in the intervention group given 100 mg / kg NAD + in the skin lesions were significantly improved, and the fibrosis level was significantly reduced.

[0035] Example 2 NAD + In vitro experimental study on inhibiting glycolysis and reducing lactic acid production and extracellular matrix synthesis

[0036] (1) Isolation and culture of keloid primary fibroblasts

[0037] After obtaining the consent of the patient and signing the informed consent form, skin lesion tissue samples were obtained from diagnosed keloid patients using aseptic techniques. The excised keloid tissue was placed in sterile physiological saline containing antibiotics to prevent bacterial infection. Subsequently, the tissue samples were minced and digested with collagenase (1 mg / mL) at 37°C for 6 hours. After centrifugation, the cell pellet was collected, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS), inoculated into culture dishes, and cultured in an incubator at 37°C and 5% CO2. When the cells grew to 80% confluence, subculture was performed, and the 3rd - 5th generation cells were used for subsequent experiments.

[0038] (2) Determination of lactic acid production and extracellular matrix synthesis

[0039] Periodic mechanical stretching (20% strain, 1 Hz frequency) was applied to keloid primary fibroblasts to simulate the mechanical environment in keloids. The cells were divided into a control group and an NAD+ treatment group (100 μM), an Eugenol treatment group (2 μM), and a Sanguinarine treatment group (2 μM). Cell supernatants and cell samples were collected at 12 h, 24 h, 36 h, and 48 h after mechanical stretching.

[0040] Figure 5 This is a line graph showing lactic acid production of keloid primary fibroblasts in the control group and the drug group at 12 h, 24 h, 36 h, and 48 h after mechanical stretching. The results show that the lactic acid production in the NAD+ drug group was significantly lower than that in the control group, indicating that NAD+ can inhibit glycolysis and reduce lactic acid production. Figure 6Protein expression diagrams of pan - lactylated proteins, H3K18la, LDHA, and COL1A1 in keloid primary fibroblasts of the control group and the drug group after mechanical stretching. The results showed that the expression levels of pan - lactylated proteins, H3K18la, and COL1A1 in the NAD+ drug group were significantly decreased, indicating that NAD+ could inhibit lactylation modification and extracellular matrix synthesis.

[0041] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, based on the present invention, some modifications and improvements can be made. Therefore, any modification or improvement made without departing from the spirit of the present invention falls within the scope of protection required by the present invention.

Claims

1. Use of nicotinamide adenine dinucleotide in the preparation of a drug for treating keloid, characterized in that, The nicotinamide adenine dinucleotide is the only active ingredient in the drug.

2. The application according to claim 1, characterized in that, The drug is used for topical skin administration.

3. The application according to claim 1, wherein The dosage forms of the drug include ointment, gel, patch, emulsion or injection solution.

4. The application according to claim 1, wherein The drug comprises nicotinamide adenine dinucleotide and excipients.

5. The application according to claim 1, wherein The nicotinamide adenine dinucleotide can inhibit the glycolysis of keloid fibroblasts.

6. The application according to claim 1, characterized in that, The nicotinamide adenine dinucleotide can reduce lactic acid production and extracellular matrix synthesis.

7. The application according to claim 1, wherein In the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 50 - 150 mg / kg.

8. The application according to claim 1, wherein In the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 100 mg / kg.

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