Use of d-tryptophan in skin wound healing

Abstract

The application provides application of D-tryptophan in skin wound healing, and belongs to the technical field of biological medicine.The application provides application of D-tryptophan (D-Trp) or a composition containing D-Trp in preparation of a skin wound healing medicament, the hemolytic activity of the D-Trp is extremely low, and the D-Trp is not prone to causing red blood cell rupture and dissolution of the human body; the cytotoxic effect of the D-Trp on a HaCat cell line is extremely small.In addition, the D-Trp can actively promote cell migration; meanwhile, the D-Trp can be combined with HIF-1a, so that the activity of genes regulating blood vessel dilation, cell division and other important wound healing processes is changed.The application also verifies the effectiveness of D-Trp in promoting wound healing in a diabetic animal model and a non-diabetic animal model by using an animal model.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of D-tryptophan in skin wound healing. Background Technology

[0002] The skin is a vital organ, playing crucial roles in regulating body temperature, preventing infection and fluid loss, and supporting immunity and sensory functions. Extensive skin damage can occur due to trauma, abrasions, ulcers, and burns. Skin injuries are prone to bacterial infection, fluid loss, and various complications. Wound dressings are typically used to protect wounds, prevent infection and dehydration, maintain a moist environment conducive to treatment, improve treatment effectiveness, and promote wound healing.

[0003] There is currently no universally accepted definition for refractory wounds. They can be caused by various factors such as trauma, infection, tumors, or nodule rupture, and there is no universally accepted definition regarding their shape, size, and depth. Generally, refractory wounds are considered chronic wounds caused by various factors that have not healed or show no tendency to heal after four weeks of standard treatment. With the aging population, the number of people with "hyperglycemia, hypertension, and hyperlipidemia" is increasing, and the number of patients with related refractory wounds is rising year by year. Although refractory wounds do not pose an immediate threat to a patient's life, they significantly impact their physical and mental health and quality of life, and increase the financial burden on their families. While various clinical methods exist for treating refractory wounds, traditional treatments are often ineffective for complex cases. Therefore, seeking new and effective methods to promote the healing of refractory wounds is an important research topic requiring collaboration across multiple disciplines. Summary of the Invention

[0004] This invention provides the application of D-tryptophan in skin wound healing, wherein the D-tryptophan can be used to treat both diabetic and non-diabetic wounds and promote effective repair of tissues in diabetic patients.

[0005] This invention provides the use of D-tryptophan or a composition containing D-tryptophan in the preparation of skin wound repair agents.

[0006] In one specific embodiment of the present invention, the skin wound includes wounds of diabetic patients and wounds of non-diabetic patients.

[0007] In one specific embodiment of the present invention, the repair effect of the skin wound repair drug includes at least one of the following: (1) promoting cell migration;

[0008] (2) Binds to HIF-1α protein;

[0009] (3) Altering the activity of various genes during wound healing;

[0010] (4) Reduce the infiltration of inflammatory cells at the wound site.

[0011] In one specific embodiment of the present invention, the genes in the wound healing process include genes related to vasodilation, cell division and other important wound healing processes.

[0012] The present invention also provides a wound-healing composition comprising D-tryptophan or a composition containing D-tryptophan.

[0013] The present invention also provides a skin wound repair agent, the active ingredient of which includes D-tryptophan or a composition containing D-tryptophan, and pharmaceutically acceptable excipients.

[0014] In one specific embodiment of the present invention, when the skin wound repair agent exists in liquid form, the content of D-tryptophan is not less than 5 μg / mL.

[0015] In one specific embodiment of the present invention, the liquid form includes using physiological saline or PBS as a solvent.

[0016] Beneficial Effects: This invention provides the application of D-tryptophan (D-Trp, HD-Trp-OH) or compositions containing D-Trp in skin wound healing. In the examples, a hemolytic activity test was conducted using 100 μg / mL, revealing a hemolysis rate of less than 2%, indicating extremely low hemolytic activity and minimal likelihood of causing rupture and lysis of human erythrocytes. This invention also used the CCK-8 assay to evaluate the cytotoxicity of D-Trp to HaCat cell lines, demonstrating that the toxicity of D-Trp at a concentration of 100 μg / mL was less than 1%, indicating minimal cytotoxicity of D-Trp to HaCat cells. Furthermore, D-Trp actively promotes cell migration; simultaneously, D-Trp can bind to HIF-1a, thereby altering the activity of genes regulating vasodilation, cell division, and other important wound healing processes. This invention also used animal models to verify the effectiveness of D-Trp in promoting wound healing in diabetic and non-diabetic animal models. Attached Figure Description

[0017] Figure 1 The graph shows the hemolysis rate results for different concentrations of D-Trp;

[0018] Figure 2 Cell viability of HaCat cells under different concentrations of D-Trp;

[0019] Figure 3 The figure shows the effect of D-Trp on HaCaT cell migration. In the figure, A: the effect of D-Trp on HaCaT cell migration at different time points, with a scale bar of 650 μm; B: the quantification of the number of HaCaT cells that migrated after treatment.

[0020] Figure 4 For use A real-time monitoring graph of the interaction between D-Trp and HIF-1α using the BLI system, with a binding constant of 5.49 × 10⁻⁶. -6 M;

[0021] Figure 5 The figure shows the evaluation results of D-Trp on wound healing in non-diabetic patients. Figure A: Schematic diagram of the treatment process and wound healing progress; B: Quantitative results of wound healing process on day 0 and day 7; C: Representative images of non-diabetic wounds treated with phosphate-buffered saline (PBS), vascular endothelial growth factor (VEGF), and D-tryptophan (D-Trp); D: Percentage of wound closure in each group at three time points; E: Representative images of heme and eosin (H&E) staining, highlighting blood vessels, with black arrows indicating newly formed blood vessels and hair follicles (scale bar: 100 μm and 20 μm). Data are expressed as mean ± standard error (SE). The sample size was n = 5. Statistical significance is as follows: ns; p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0022] Figure 6 The following is an assessment of D-Trp on in vivo diabetic wound healing: A: Schematic diagram of treatment process and wound healing progress; B: Quantitative graph of wound healing process on days 0, 7, and 14; C: Representative image of chronic diabetic wounds treated with phosphate-buffered saline (PBS), vascular endothelial growth factor (VEGF), and D-tryptophan (D-Trp); D: Percentage of wound closure in each group at three time points; E: Representative image of heme and eosin (H&E) staining, highlighting blood vessels, with black arrows indicating newly formed blood vessels and hair follicles (scale bar: 100 μm and 20 μm); Plasma biochemical parameters after 14 days of treatment: F: glucose level; G: aspartate aminotransferase; H: triglycerides; I: total cholesterol; Data are expressed as mean ± standard error (SE), with a sample size of n = 6. Statistical significance is as follows: NS; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Detailed Implementation

[0023] This invention provides the use of D-tryptophan or a composition containing D-tryptophan in the preparation of skin wound repair agents.

[0024] The D-Trp described in this invention is the enantiomer of natural L-tryptophan. L-tryptophan mainly participates in protein synthesis, while D-tryptophan has unique properties that may be beneficial for wound repair in diabetic patients. D-Trp is a white to off-white solid with a molecular weight of 204.23. This invention does not specifically limit the source of the D-Trp; for example, in the examples, it was purchased from MCE (MedChemExpress) with a purity of 99.97%.

[0025] In one specific embodiment of the present invention, the skin wound includes wounds from diabetic patients and wounds from non-diabetic patients. In one embodiment of the present invention, the migration ability of HaCaT cells treated with D-Trp was measured. The results showed that cells treated with D-Trp migrated significantly faster when crossing the wound area, and were able to more quickly and effectively close the gaps; that is, D-Trp treatment can significantly enhance the migration ability of HaCaT cells.

[0026] In another embodiment of the present invention, using The BLI system was used to detect the binding affinity of D-Trp to HIF-1α protein. Since HIF-1α is a major regulator of gene expression in the wound microenvironment under hypoxic conditions, D-Trp may alter the activity of genes that regulate vasodilation, cell division, and other important wound healing processes by binding to HIF-1α. In other words, D-Trp can bind to HIF-1α, thereby potentially altering its activity and function in wound healing.

[0027] In another embodiment of the present invention, a non-diabetic mouse model with severe skin damage was constructed, and it was found that the wound healing speed was significantly accelerated after D-Trp treatment. The formation and re-epithelialization of granulation tissue were significantly increased after using D-Trp, and the number of inflammatory cells entering the wound was also reduced.

[0028] In another embodiment of the present invention, a diabetes model was constructed to verify the effect of D-Trp on improving wound healing in diabetic patients. After treatment with D-tryptophan, similar results as in non-diabetic patients were achieved, with a significant increase in the formation and re-epithelialization of granulation tissue and a reduction in inflammatory cell infiltration at the wound site, indicating the effectiveness of D-Trp in promoting wound healing.

[0029] The present invention also provides a wound-healing composition comprising D-tryptophan or a composition containing D-tryptophan.

[0030] The wound-healing composition of this invention can be prepared as a topical medication comprising an active layer, a support layer, and an adhesive layer. The active layer can be a nanofiber membrane loaded with D-tryptophan or a hydrogel layer containing D-tryptophan. The nanofiber membrane can be prepared by electrospinning, where D-tryptophan is mixed with a spinnable polymer solution and then electrospun to form a nanofiber structure, which slowly releases D-tryptophan. The preparation method of the hydrogel layer containing D-tryptophan is similar to that of the gel-type composition. The support layer of this invention is generally a material with certain strength and breathability, such as non-woven fabric or polyurethane membrane. Its function is to provide support and fixation for the active layer, facilitating the use and handling of the dressing. If the dressing needs to adhere to the skin, an adhesive layer, such as a layer containing medical pressure-sensitive adhesive, can be provided on one side. Release paper can be placed on the adhesive layer; the release paper can be peeled off for application.

[0031] The present invention also provides a skin wound repair agent, the active ingredient of which includes D-tryptophan or a composition containing D-tryptophan, and pharmaceutically acceptable excipients.

[0032] In one specific embodiment of the present invention, when the skin wound repair agent exists in liquid form, the D-tryptophan content is not less than 5 μg / mL. In another specific embodiment of the present invention, the liquid form includes using physiological saline or PBS as a solvent.

[0033] To further illustrate the present invention, the application of D-tryptophan provided by the present invention in skin wound healing is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Hemolysis Analysis

[0035] First, blood was collected from healthy individuals and treated with an anticoagulant solution. Next, the treated blood was rinsed twice with physiological saline and then resuspended to achieve a cell concentration of 102. 7 ~10 8 cells / mL. The diluted red blood cell suspension was mixed with D-Trp samples dissolved in physiological saline. The mixture was incubated at 37°C for 30 minutes, followed by centrifugation at 1000 rpm for 5 minutes. The supernatant was analyzed at a wavelength of 540 nm. A negative control was prepared using physiological saline, while a positive control was prepared using Triton X-100. The percentage of hemolysis was calculated using the following formula:

[0036] Hemolysis percentage H% = (Sample A - Negative Control A) / Positive Control A × 100%.

[0037] The results are as follows Figure 1As shown, when the concentration of D-Trp is 100 μg / mL, its hemolysis rate is less than 2%. This indicates that its hemolytic activity is extremely low, and it is unlikely to cause rupture and lysis of human red blood cells, thus posing a harm to the human body.

[0038] Example 2 Cytotoxicity Analysis

[0039] When evaluating the cytotoxicity of the HaCat cell line using the CCK-8 assay, the cells were first cultured in an appropriate growth medium until they reached 70%–80% confluence in the tissue culture flasks. Subsequently, the cells were isolated using trypsin and counted using a hemocytometer. Approximately 1 × 10⁶ cells were cultured per well. 4 Up to 1×10 5 Cells were seeded into 96-well plates and allowed to adhere for 24 hours. After cell adhesion, serial dilutions of D-Trp treatment were prepared in growth medium, and these dilutions were used to replace the medium in the wells. Control wells containing untreated cells and control wells containing a known cytotoxic agent were set up for comparison. The plates were incubated in a CO2 incubator at 37°C for 24 hours. After incubation, CCK-8 solution was added to each well according to the manufacturer's instructions, and the plates were incubated for another 1–4 hours to induce a colorimetric reaction.

[0040] Next, absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as a percentage using the following formula: Cell viability (%) = (Sample A / Control A) × 100%, where Sample A represents the absorbance of treated cells and Control A represents the absorbance of untreated control cells. The cytotoxic effect of D-Trp on HaCat cells was evaluated using the results.

[0041] The results are as follows Figure 2 As shown, the toxicity of D-Trp at a concentration of 100 μg / ml is less than 1%, and at this concentration, D-Trp has minimal cytotoxic effect on HaCat cells.

[0042] Example 3: In vitro wound healing analysis

[0043] HaCat cells were placed in 12-well plates. When the cell density reached 80%–90%, culture medium (as a control group), VEGF (20 ng / ml), and D-tryptophan (20 μg / ml) were added. Two hours later, the cell layer of each culture plate was scraped with the tip of a sterile pipette to create scratch wounds. Cell migration distance was calculated using ImageJ software (1.47v; National Institutes of Health, Bethesda, MD, USA). Cell images were taken using a microscope at time intervals of 6, 12, 24, and 36 hours. The experiment was repeated three times.

[0044] The results are as follows Figure 3 As shown, compared with the control group, D-Trp treatment significantly enhanced the migration ability of HaCaT cells. Figure 3 (See A and C). Specifically, under the experimental conditions of Example 3, this invention observed that cells treated with D-Trp migrated significantly faster when crossing the wound area, and were able to close the gap more quickly and effectively than control cells. In contrast, the migration rate of the control group was relatively slow, indicating that D-Trp plays an important role in promoting cell migration, a key process in wound closure. This demonstrates that D-Trp can actively promote cell migration, and since cell migration is a key step in wound healing, it further highlights its potential therapeutic value in accelerating tissue repair.

[0045] Example 4: Binding Affinity

[0046] use The BLI system was used to detect the binding affinity of D-tryptophan to HIF-1α protein.

[0047] (1) Prepare a D-tryptophan solution with a concentration between 2.5 mM and 0.15625 mM.

[0048] (2) Dilute the biotinylated HIF-1α to 10 μg / ml to immobilize it. Then add streptavidin to the biosensor.

[0049] (3) Use Following the manufacturer's instructions, load streptavidin onto the SA biosensor using the BLI system. Incubate the streptavidin-loaded biosensor in HIF-1α solution for a sufficient time to allow the receptor to bind to streptavidin. Rinse the biosensor with detection buffer.

[0050] (4) Place the biosensor in the reference buffer for a period of time to establish a baseline signal.

[0051] (5) The biosensor was transferred to a solution containing different concentrations of D-tryptophan. The ligand was then allowed to bind to immobilized HIF-1α for 300 seconds.

[0052] (6) Transfer the biosensor back to the reference buffer and measure the dissociation of D-tryptophan and HIF-1α within 150 seconds.

[0053] In the above experiments, this invention also included appropriate control groups, such as biosensors containing only streptavidin (without receptor) or biosensors with receptors but no ligands. The results indicate that D-Trp can bind to HIF-1a, potentially altering its activity and function in wound healing. Figure 4This discovery is crucial because HIF-1a is a major regulator of gene expression under hypoxic conditions commonly found in the wound microenvironment. By binding to HIF-1a, D-Trp may alter the activity of genes regulating vasodilation, cell division, and other important wound healing processes.

[0054] Example 5: Wound healing effect of D-Trp on normal and diabetic mouse skin injury models

[0055] To evaluate the healing effect of D-Trp, a 6 mm diameter wound was placed on the dorsal side of C57BL / 6J mice. This study used 15 male normal C57BL / 6J mice and 15 male C57BL / 6J diabetic-induced mice, aged 10–12 weeks. All mice were randomly assigned to one of three groups: control (PBS), VEGF, or D-Trp. Five mice were assigned to each group. All mice were acclimatized for one week prior to surgery. All surgeries were performed under sterile conditions. After standard anesthesia with an intraperitoneal injection of 2% pentobarbital (4–4.5 μL per 20 g body weight), the back hair above the tail and below the back was removed with depilatory cream to prepare for surgery. A circular wound with a diameter of 6 mm and full skin thickness was created using a needle biopsy. After removing the wound skin, the control group received 30 μL of PBS, while the VEGF and D-Trp groups received a final concentration of 5 μg / mL and 5 μg / mL, respectively, without any bandaging. All tissues were collected from five mice in each of the three groups in both normal and diabetic model mice on days 7 and 14. Wound area was monitored in both groups on days 1, 3, and 7, and on days 1, 7, and 14. Medication was applied topically to the wounds daily using a pipette. Wounds were monitored daily for signs of infection or other complications. All mice underwent standard anesthesia, and the wound area was recorded using a camera (E-M10, Olympus), with the wound boundaries traced and measured using ImageJ software.

[0056] In vivo studies in a non-diabetic mouse model with severe skin lesions showed that the wound healing rate in the D-Trp group was significantly faster than that in the control group. Figure 5(C and D). All groups showed wound contraction and the formation of a wound bed supported by new tissue, indicating an overall healing response. However, on days 3 and 7, the D-Trp group showed faster wound area contraction and thicker wound tissue compared to other groups, based on lesion area and tissue thickness. Notably, on day 3, the D-Trp group shed a significant amount of new skin tissue and new white hair. This indicates increased epithelialization and hair follicle regeneration. On day 7, the D-Trp group was the first to form new epidermis and grow hair around it, indicating more thorough and rapid wound healing. H&E staining of skin sections showed a significant improvement in wound healing compared to the control group. Figure 5 H&E staining showed that the use of D-tryptophan significantly increased the formation and re-epithelialization of granulation tissue, and reduced the number of inflammatory cells entering the wound.

[0057] Example 6: A diabetic model of improved wound healing using D-Trp

[0058] A 14-day experiment was conducted using streptozotocin (STZ)-induced diabetic mice. Figure 6 (A). The results showed that, compared with the control group, a concentration of 5 μg / ml of D-tryptophan (D-Trp) significantly promoted wound healing in the diabetic model. Figure 6 (B) Although all groups showed wound contraction and new tissue formation, the D-Trp group showed faster wound area reduction and thicker tissue formation. Figure 6 (C) This indicates that the healing process of D-Trp is more effective and thorough. Notably, the D-Trp group showed early signs of epithelialization and tissue regeneration, with significant collapse of newly formed tissue and growth of new hair. Figure 6 (C) By day 14, the D-Trp group showed the most comprehensive healing response, with complete recovery of epidermal and surrounding hair growth. These results highlight the potential of D-Trp as a promising therapeutic agent in accelerating and improving wound healing. H&E staining of skin sections showed that the wound healing capacity of the D-tryptophan treatment group was significantly enhanced compared to the control group. Figure 6 (E). Specifically, treatment with D-tryptophan significantly increased granulation tissue formation and re-epithelialization, while reducing inflammatory cell infiltration at the wound site. Notably, these results were comparable to those observed in the non-diabetic control group, demonstrating the effectiveness of D-tryptophan in promoting wound healing.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of D-tryptophan or D-tryptophan-containing compositions in the preparation of skin wound repair agents.

2. The application according to claim 1, characterized in that, The skin wounds include those of diabetic patients and those of non-diabetic patients.

3. The application according to claim 1, characterized in that, The repairing effect of the skin wound repair drug includes at least one of the following: (1) promoting cell migration; (2) Binds to HIF-1α protein; (3) Altering the activity of various genes during wound healing; (4) Reduce the infiltration of inflammatory cells at the wound site.

4. The application according to claim 3, characterized in that, The genes mentioned in the wound healing process include those related to vasodilation, cell division, and other important wound healing processes.

5. A wound-healing composition, characterized in that, Including D-tryptophan or compositions containing D-tryptophan.

6. A skin wound repair agent, characterized in that, The active ingredient includes D-tryptophan or a composition containing D-tryptophan, and pharmaceutically acceptable excipients.

7. The skin wound repair agent according to claim 6, characterized in that, When the skin wound repair agent is in liquid form, the content of D-tryptophan is not less than 5 μg / mL.

8. The skin wound repair agent according to claim 7, characterized in that, The liquid form includes those using physiological saline or PBS as solvents.

Images