Application of nicotinamide adenine dinucleotide in preparation of medicine for treating keloid

Abstract

The invention discloses an application of nicotinamide adenine dinucleotide in preparation of a medicine for treating keloid, and by researching the pathophysiological mechanism of keloid, it is found that mechanical related fibroblasts of keloid patients have metabolic reprogramming changes. Fibroblast populations which are mainly pathogenic to keloid have metabolic reprogramming caused by mitochondrial dysfunction, especially glycolysis level enhancement in cells and lactic acid accumulation. The medicine, namely NAD +, capable of regulating and controlling keloid fibroblast glycolysis and mitochondrial metabolism is found and verified through cell and animal experiments, and NAD + inhibits development of keloid by improving mitochondrial dysfunction, reducing lactic acid accumulation and inhibiting fibroblast migration and collagen synthesis. According to the invention, NAD +, which is a common medicine with good safety, is applied to keloid for the first time, and the scheme has small toxic and side effects and also has positive effects on oxidation resistance, aging resistance and cancer resistance, so that a new scheme is provided for treating keloid by medicines.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of nicotinamide adenine dinucleotide in the preparation of a drug for treating keloid. Background Art

[0002] Keloid is a fibrous dysproliferative disease of the skin 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 irregular edges and pigmentation. The growth of keloids not only causes changes in the appearance of the skin, but is also usually accompanied by itching, pain and other discomfort. These symptoms often have a negative impact on the patient's spirit and mood, thus affecting their daily life and social activities. Studies have shown that the presence of keloids is closely related to the patient's quality of life. Patients often feel inferior and anxious, leading to mental health problems.

[0003] There are many treatments for keloids, and 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 achieve certain improvements after surgery, the recurrence rate is relatively high, which often makes the effectiveness of surgery questionable. 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 course of hormone therapy is long, and the effect is often delayed, with a high clinical recurrence rate and many adverse reactions. In addition, radiotherapy and chemotherapy have shown certain effects in reducing lesions, but due to its large side effects, it is limited in clinical application, especially in young patients or patients with liver and kidney dysfunction, which may cause damage to their normal tissues.

[0004] Although there are various treatments for keloids at present, there are still insufficient preventive measures for their occurrence or recurrence, which makes patients still face a high risk of recurrence after the treatment. Therefore, it is particularly important to explore preventive drugs for keloids. The development of such drugs can not only reduce the incidence of keloids, but also delay the further development of keloids that have already formed, 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 patients with keloids, and ultimately achieve individualized and comprehensive health management. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide an application of nicotinamide adenine dinucleotide in the preparation of a drug for treating keloids. Nicotinamide adenine dinucleotide can inhibit the occurrence and development of keloids by inhibiting glycolysis of keloid fibroblasts, reducing lactic acid production and extracellular matrix synthesis.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: The present invention provides application of nicotinamide adenine dinucleotide in preparing a drug for treating keloid.

[0007] In order to explore drug targets, we carefully explored the pathophysiological mechanism of keloids. In order to explore effective new drugs, we carefully studied the pathophysiological mechanism of keloids. In the study population, we found that mechanical-related fibroblasts in patients with keloids had metabolic reprogramming changes. Further animal and cell experiments proved that one of the pathogenesis of keloid development is that under tensile stimulation, mitochondrial dysfunction of keloid fibroblasts induces increased LDHA expression, causing lactic acid accumulation, thereby promoting fibroblast proliferation and enhanced myofibrosis.

[0008] Given our findings that mechanically related mitochondrial dysfunction and enhanced glycolytic capacity promote the development of keloids, we screened 404 FDA-approved compounds related to mitochondria and glycolysis to identify potential therapeutic agents. We cultured primary keloid fibroblasts for high-throughput drug screening. High-content cell imaging was used to evaluate the effects of these compounds on the expression of LDHA and COL1A1 in fibroblasts after tensile stimulation. During the drug screening, we found a drug, nicotinamide adenine dinucleotide (NAD + ) can significantly reduce the expression of LDHA and COL1A1. Further cell and animal experiments have shown that NAD + By inhibiting the glycolysis of keloid fibroblasts, reducing lactic acid production and extracellular matrix synthesis, inhibiting the occurrence and development of keloid (such as Figure 1 as shown).

[0009] Preferably, the drug is for topical administration to the skin.

[0010] Preferably, the dosage form of the drug includes ointment, gel, patch, emulsion or injection.

[0011] Preferably, the drug comprises nicotinamide adenine dinucleotide and excipients.

[0012] Preferably, the nicotinamide adenine dinucleotide can inhibit glycolysis of keloid fibroblasts.

[0013] Preferably, the nicotinamide adenine dinucleotide is capable of reducing lactate production and extracellular matrix synthesis.

[0014] NAD +It is an important coenzyme in redox reactions, and its decreased level is closely related to the occurrence of aging and fibrotic diseases. + It plays a central role in maintaining cell metabolism, energy production, and gene repair. + At present, it has a wide range of clinical application prospects, involving aging, kidney disease, cardiometabolic disease, neurodegenerative disease, inflammation, fibrosis and cancer. In vitro experiments have shown that NAD + It has the effects of inhibiting glycolysis, reducing lactic acid production, inhibiting collagen synthesis and anti-fibrosis.

[0015] Preferably, in the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 50-150 mg / kg.

[0016] Preferably, in the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 100 mg / kg.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention studies the pathophysiological mechanism of keloids and finds that the mechanically related fibroblasts of patients with keloids have metabolic reprogramming changes. Our research found that the main pathogenic fibroblast population of keloids has metabolic reprogramming caused by mitochondrial dysfunction, especially the enhancement of intracellular glycolysis and accumulation of lactic acid. The present invention searches for and verifies the drug that can regulate the glycolysis and mitochondrial metabolism of keloid fibroblasts through cell and animal experiments - NAD + , NAD + By improving mitochondrial dysfunction, reducing lactic acid accumulation, inhibiting fibroblast migration and collagen synthesis, the development of keloids is inhibited. + This common drug with good safety is used to treat keloids. It has few toxic side effects and also has positive effects on antioxidant, anti-aging and anti-cancer effects, providing a new option for the drug treatment of keloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 NAD + Mechanism of action diagram; Figure 2 The picture shows the situation of nude mice in the control group and the drug-treated group on the 21st day after modeling; Figure 3 The pictures are of keloid nodules in nude mice in the control group and the drug-treated group on the 21st day after modeling; Figure 4 The growth curves of keloid nodules in nude mice of the control group and the drug-treated group are shown; Figure 5 Line graphs showing lactate generation after mechanical stretching of primary keloid fibroblasts in the control group and drug group; Figure 6 This is a graph showing the degree of lactic acidification and extracellular matrix synthesis proteins in primary keloid fibroblasts after mechanical stretching in the control group and the drug group. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0020] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0021] Example 1 NAD + In vitro experimental study on reducing the fibrosis degree of keloid (1) Establishment of humanized keloid mouse model and drug administration After the patient agreed and signed the informed consent form, skin lesion tissue samples were obtained from the confirmed keloid patients after local anesthesia using sterile technology. The excised keloid tissue was placed in sterile saline containing antibiotics to prevent bacterial infection, followed by centrifugation, washing and cutting to prepare 1 mm tissue blocks.

[0022] Immunodeficient nude mice were selected and 1% sodium pentobarbital was administered at 50 mg / kg -1 After anesthesia, prepare a surgical incision under the back of the mouse, clean and disinfect the surgical area, transplant the treated keloid tissue into the subcutaneous tissue of the mouse, suture the skin incision with a fine needle and suture, and construct a humanized keloid mouse model. One week after modeling, keloid-like nodules were observed on the back of the modeled mice, indicating that the modeling was successful. Twelve mice with successful modeling were given 50mg / kg, 100mg / kg, and 150mg / kg NAD + The injection was continued for 21 days. In addition, 3 mice in the control group were not treated. A total of 15 mice were given regular feed and drinking water.

[0023] (2) Determination of relative scar inhibition rate The keloid nodule areas of each nude mouse were measured at 0, 5, 10, and 15 days after administration, and the keloid nodule conditions were recorded using a camera at a fixed height, and the keloid nodule growth curve was drawn.

[0024] Figure 2 The following are the conditions of nude mice in the control group and the drug-treated group on the 21st day after modeling, where the amount of yoda1 (a specific activator of piezo1 that increases environmental mechanical responsiveness) added was 1 mg / kg every other day; Figure 3 The pictures are of keloid nodules in nude mice in the control group and the drug-treated group on the 21st day after modeling; Figure 4The figure is the growth curve of keloid nodules in nude mice of the control group and the drug-treated group. Figure 3 and Figure 4 The results showed that compared with the control group, 100 mg / kg NAD was administered intralesionally. + The growth rate, volume and quality of nodules in the intervention group mice were significantly improved, and the fibrosis level was significantly reduced.

[0025] Example 2 NAD + In vitro experimental study on inhibition of glycolysis and reduction of lactic acid production and extracellular matrix synthesis (1) Isolation and culture of primary keloid fibroblasts After the patient agreed and signed the informed consent form, skin lesion tissue samples were obtained from confirmed keloid patients using aseptic techniques. The excised keloid tissue was placed in sterile 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. The cell pellets were collected after centrifugation, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS), inoculated in a culture dish, and cultured in an incubator at 37°C and 5% CO2. When the cells grew to 80% confluence, they were subcultured and the 3rd to 5th generation cells were used for subsequent experiments.

[0026] (2) Determination of lactate production and extracellular matrix synthesis Cyclic mechanical stretching (20% strain, 1 Hz frequency) was applied to primary keloid fibroblasts to simulate the mechanical environment in keloid. The cells were divided into control group, NAD+ treatment group (100 μM), Eugenol treatment group (2 μM), and Sanguinarine treatment group (2 μM), and cell supernatants and cell samples were collected 12 h, 24 h, 36 h, and 48 h after mechanical stretching.

[0027] Figure 5 The figure shows the line graph of lactate production in primary keloid fibroblasts in the control group and drug group at 12 h, 24 h, 36 h and 48 h after mechanical stretching. The results showed that lactate production in the NAD+ drug group was significantly lower than that in the control group, indicating that NAD+ can inhibit glycolysis and reduce lactate production. Figure 6 The following are protein expressions of pan-lactated proteins, H3K18la, LDHA, and COL1A1 in primary fibroblasts of keloid in the control group and drug group after mechanical stretching. The results showed that the expression levels of pan-lactated proteins, H3K18la, and COL1A1 in the NAD+ drug group were significantly reduced, indicating that NAD+ can inhibit lactic acid modification and extracellular matrix synthesis.

[0028] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. Application of nicotinamide adenine dinucleotide in the preparation of drugs for treating keloids.

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

3. The use according to claim 1, characterized in that: The dosage form of the drug includes ointment, gel, patch, emulsion or injection.

4. The use according to claim 1, characterized in that: The medicine comprises nicotinamide adenine dinucleotide and excipients.

5. The use according to claim 1, characterized in that: The nicotinamide adenine dinucleotide can inhibit the glycolysis of keloid fibroblasts.

6. The use according to claim 1, characterized in that: The nicotinamide adenine dinucleotide can reduce lactate production and extracellular matrix synthesis.

7. The use according to claim 1, characterized in that: In the drug for treating keloid, the concentration of nicotinamide adenine dinucleotide is 50-150 mg / kg.

8. The use according to claim 1, characterized in that: In the keloid treatment drug, the concentration of nicotinamide adenine dinucleotide is 100 mg / kg.

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