Application of Euproctus aschimbons skin wound healing in the process of molting

Abstract

The application belongs to the technical field of biological medicine and wound repair, and particularly relates to application of Anji small salamander in skin wound healing. The application has good ability of promoting wound healing by using the biological active substances in the ecdysis of Anji small salamander, can effectively improve the skin wound healing efficiency, shorten the exuviation time, inhibit the scar formation in the wound healing process, promote the regeneration of skin appendages in the wound healing process and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and wound repair technology, specifically involving the application of active substances in the skin of the Anji salamander (Hynobius amjiensis) in skin wound healing, accelerating the repair of deep skin damage, promoting tissue regeneration and inhibiting the formation of pathological scars. Background Technology

[0002] As the largest organ and primary physical barrier in the human body, the skin, composed of the epidermis, dermis, and appendages, is frequently subjected to various injuries, such as abrasions, cuts, burns, scalds, and sunburns. Wound healing can be divided into primary healing and secondary healing. Primary healing is a simple healing process that occurs in sterile wounds with smooth edges, such as surgical wounds, and typically heals within 6-8 days. Secondary healing begins when there is significant tissue loss or infection complications, leading to the formation of granulation tissue and scarring. It takes longer to heal, carries a higher risk of infection, and has a poorer healing outcome.

[0003] Skin injury repair is a complex process involving four overlapping, ordered phases: hemostasis, inflammation, proliferation, and remodeling. When skin is injured, hemostasis is activated to stop bleeding, followed by an inflammatory response to clear invading pathogens and prepare the tissue for repair. During the proliferation phase, angiogenesis, fibroblast migration, and re-epithelialization occur. In the remodeling phase, granulation tissue is replaced by scar tissue. In healthy individuals and when the injury does not extend into the deep dermis, the skin can usually heal effectively through spontaneous regeneration. However, injuries deep in the dermis heal significantly more slowly and are often accompanied by pathological skin contraction and permanent scarring. Therefore, developing innovative therapies that accelerate deep wound healing and reduce scarring is a critical challenge that urgently needs to be addressed in the medical field.

[0004] Currently, commonly used clinical medications for promoting healing include Kangfuxin Liquid (based on American cockroach extract), growth factor preparations, and various topical Chinese medicine preparations, which are widely used in the treatment of wounds such as trauma, ulcers, burns, and scalds. Although these medications can accelerate epithelialization of wounds to some extent, they still have significant limitations in promoting dermal structural reconstruction, reducing disordered collagen deposition, and inhibiting pathological scarring. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned limitations of current wound healing medications by developing novel drugs that promote rapid healing of deep skin injuries and inhibit scar formation through the molting of the Anji salamander. Solving these technical problems not only provides new options for clinical treatment but also brings better treatment outcomes and improved quality of life to patients.

[0006] In a first aspect, the present invention provides the application of the molting of the Anji salamander in the healing of skin wounds.

[0007] The Anji salamander (scientific name: *Hynobius amjiensis*) is an amphibian belonging to the family Hynobiidae and the genus *Hynobius*. It is relatively small, with males measuring approximately 153-166 mm in total length and females around 166 mm. The head is oval and flattened, with its length slightly greater than its width. The trunk is stout and slightly flattened. The tail base is nearly round, gradually flattening laterally towards the rear. The dorsal skin is smooth, with a longitudinal groove running from behind the eye to the neck fold. The limbs are relatively slender; when the fore and hind limbs are close together, the fingers and toes overlap or reach the palms and soles. The male salamander has a longitudinally slit anal opening with a small papilla in the center of the anterior margin. The eggs are round, black on the animal pole and grayish-white on the vegetal pole; the egg diameter is 3.5 mm, and including the transparent capsule, the total diameter is 12-14 mm.

[0008] While existing technologies have revealed the significant potential of biological resources in wound healing, some studies have also demonstrated their potential. For example, extracts of the mucus from the Chinese giant salamander (Andrias davidianus) have been shown to be effective in wound healing (CN106511399A). Planarian extracts have been shown to promote wound healing (CN120267707A).

[0009] However, there is currently little research on the Anji salamander resource, and no function of the Anji salamander resource in wound healing has been proposed, especially as the shed skin of the Anji salamander.

[0010] The Anji salamander exhibits a physiological characteristic of periodic molting throughout its life cycle. Its molting frequency is closely related to environmental humidity, temperature, growth stage, and reproductive cycle. In high-humidity environments, its epidermal metabolism is active, and the molting cycle is approximately 1-3 days. The molting process is passive and gradual: the old epidermis first appears with fissures around the mouth and sides of the body, and then the individual gradually sheds the entire epidermis completely by twisting its body and rubbing against vegetation. The entire process is not accompanied by tissue damage or bleeding.

[0011] The shed skin is a thin membrane that completely covers the original body outline and can be found in moss in its habitat and in winter breeding waterholes. The molting of the Anji salamander is a product of natural physiological behavior and can be obtained without human intervention or harm to the individual.

[0012] This invention is the first to discover that the molted tissue of the Anji salamander has a significant ability to promote skin wound healing, especially in accelerating the repair of full-thickness skin damage, promoting orderly collagen deposition, and inhibiting the formation of pathological scars, showing superior potential compared to existing drugs.

[0013] The skin wounds described include full-thickness skin defects, second- or third-degree burns, surgical wounds, or chronic ulcers.

[0014] Preferably, the skin wound refers to a wound that extends into the dermis.

[0015] Preferably, skin wound healing includes inhibiting the formation of pathological scars during the wound healing process, especially inhibiting the formation of pathological scars during the healing of wounds deep in the dermis. The healing process of injuries deep in the dermis is significantly slow and is often accompanied by pathological skin contraction and permanent scar formation. Although there are many drugs for skin wound healing, most leave scars, and currently available scar repair drugs are very expensive. This invention discovers that the molting of the Anji salamander has a better therapeutic effect on the healing of injuries deep in the dermis and effectively inhibits scar formation.

[0016] Ideally, skin wound healing also includes shortening the healing time.

[0017] In a second aspect, the present invention provides a medicament for promoting skin wound healing, comprising the molted skin of the Anji salamander.

[0018] A third aspect of the present invention provides a medicament for inhibiting the formation of skin wound scars, comprising the molted skin of the Anji salamander.

[0019] The drugs mentioned in the second or third aspect also include pharmaceutically acceptable carriers.

[0020] The dosage forms of the drugs mentioned in the second or third aspect are topical gels, ointments, sprays, dressings, or patches, etc.

[0021] A fourth aspect of the present invention provides a method for preparing a drug containing the molting skin of the Anji salamander, comprising the following steps:

[0022] a. Obtain the molted skin of the Anji salamander;

[0023] b. Wash 5 times with D-Hanks under ice bath conditions (0-4℃). Use MagNA Lyser fully automated tissue homogenizer to thoroughly homogenize the molted skin obtained in step a to obtain the molted skin extract of *Andrias davidianus*.

[0024] c. After sterilizing the extract of *Andrias davidianus* obtained in step b by filtration through a 0.22 μm filter membrane, freeze-dry to obtain *Andrias davidianus* molting powder.

[0025] By implementing the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0026] This study is the first to propose using the molted skin of the Anji salamander as a potential resource for promoting skin wound healing. It suggests that this method can effectively improve wound healing efficiency, shorten scab shedding time, and inhibit scar formation during the healing process, particularly for deep wound repair and scar inhibition. This innovation breaks through existing wound healing treatment approaches, providing a novel biological resource for developing new wound treatment drugs and potentially addressing the shortcomings of existing drugs in deep wound healing and scar inhibition.

[0027] The molting of the Anji salamander has the following characteristics in skin wound healing: highly efficient healing promotion, significantly shortening healing time; significant anti-scarring effect, effectively inhibiting the formation of pathological scars; high biocompatibility; and wide application, which can be developed into various topical formulations suitable for various wound types. Attached Figure Description

[0028] Figure 1 The molting of the Anji salamander;

[0029] Figure 2 shows the healing status of knife wounds in rats under different treatment groups;

[0030] Figure 3 Statistical analysis of the wound healing time of rats with knife wounds in different treatment groups;

[0031] Figure 4 HE-stained sections of whole-dermal lesions in rats from different treatment groups;

[0032] Figure 5 Masson-stained sections of rats with full-layer dermal injury corresponding to different treatment groups;

[0033] Figure 6 Statistics on collagen deposition rate on day 14 of full-thickness skin wounds;

[0034] Figure 7 The healing status of scalded wounds in rats corresponding to different treatment groups;

[0035] Figure 8 Statistical analysis of the healing time of scalded wounds in rats from different treatment groups;

[0036] Figure 9 HE-stained sections of scalded rats from different treatment groups;

[0037] Figure 10 Masson-stained sections of scalded rats from different treatment groups;

[0038] Figure 11 Statistics on collagen deposition rate in burn wounds on day 14;

[0039] Figure 12 Cells corresponding to different treatments;

[0040] Figure 13 Cell activity corresponding to different treatments. Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments, but should not be construed as limiting the present invention.

[0042] Example 1

[0043] Preparation of Anji salamander molting powder

[0044] Collecting naturally shed skins of Anji salamanders.

[0045] Wash five times with pre-cooled D-Hanks solution to remove dirt and impurities.

[0046] The molted skin was placed in a sterile tube and then placed in the MagNA Lyser fully automated tissue homogenizer. After thorough homogenization, it became completely liquid.

[0047] Transfer to centrifuge tubes, filter through a 0.22 μm filter membrane for sterilization, and collect the filtrate.

[0048] The filtrate was freeze-dried at ultra-low temperature to obtain the molted skin powder of the Anji salamander.

[0049] Dissolve the powder in ultrapure water to prepare solutions of three concentrations: 1 μg / mL, 10 μg / mL, and 100 μg / mL, for subsequent experiments.

[0050] Example 2

[0051] Efficacy evaluation of a rat model of full-thickness skin defects

[0052] 1. Establishment of a rat full-thickness skin injury model: Eight-week-old female SD rats were fed according to animal health and treatment procedures (all experimental rats in this invention were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., and the entire experimental operation complied with the "Regulations on the Management of Animal Experiments"). The rats underwent dorsal hair removal one day before modeling to observe for any allergic reactions. Rats without allergies were anesthetized by injecting 1% sodium pentobarbital at a dose of 100 μL / 10 g, according to their body weight. After disinfecting the skin on the rat's back, four symmetrical, 10 mm circular wounds were created along the midline of the back, involving the complete excision of the entire skin layer.

[0053] 2. Experimental grouping and treatment: The successfully modeled rats were randomly divided into 5 groups (n=6): (1) Negative control group: 100 μL of D-Hanks solution was applied to the wound for each treatment; (2) Positive control group: 100 μL of Kangfuxin liquid (National Drug Approval Number Z51021834) was applied to the wound for each treatment; (3) Low concentration group (1 μg / ml): 100 μL of drug prepared from the molting of Anji salamander was applied to the wound for each treatment; (4) Medium concentration group (10 μg / ml): 100 μL of drug prepared from the molting of Anji salamander was applied to the wound for each treatment; (5) High concentration group (100 μg / ml): 100 μL of drug prepared from the molting of Anji salamander was applied to the wound for each treatment.

[0054] 3. Observation indicators: (1) Recording of wound healing: Morphological changes were recorded by taking pictures with a mobile phone; (2) Histopathological analysis: Rats in each group were randomly sacrificed on the 3rd, 7th and 14th days of modeling, and skin samples were taken from the modeling site, fixed with 4% paraformaldehyde, and embedded in paraffin for sectioning. Hematoxylin-eosin (H&E) staining was performed to observe the infiltration of inflammatory cells and the proliferation of fibroblasts, and Masson trichrome staining was used to assess collagen fiber deposition and tissue repair; (3) Masson staining collagen deposition assessment on the 14th day: Image J software was used to quantitatively assess the collagen deposition stained by Masson.

[0055] Result: In Figure 2 In the study, wound healing was observed within 14 days after modeling. Compared to the positive control, the medium and high concentrations showed better healing effects at 3, 7, and 13 days, especially at 13 days, where the healing effect was significantly better than the positive control group. The high-concentration drug prepared from the molted skin of the Anji salamander showed even better results. See the healing rate statistics below. Figure 3 On day 3 of wound healing, the healing rate of all groups except the negative control was above 40%, and all concentrations of salamander molt were superior to the positive control. On day 7 of wound healing, the healing rate of all groups except the negative control was above 60%, but the healing rate of all concentrations of salamander molt was around 70%, superior to the positive control. On day 13 of wound healing, the healing rate of all groups except the negative control was above 90%. There was no significant difference between the positive control group and the low concentration group of salamander molt, but there were significant differences with the medium and high concentration groups, and the medium and high concentration groups were close to the healing state.

[0056] from Figure 4 The H&E staining results shown indicate that:

[0057] Epidermal integrity: The negative control group showed defects and discontinuities in the epidermis; the positive control group showed some improvement in epidermal condition, but still had shortcomings; the low concentration group showed limited repair effect on epidermal integrity. The medium and high concentration groups showed more complete and continuous epidermis, indicating that medium and high concentrations of drugs prepared from the molted skin of the Anji salamander are more beneficial for maintaining or repairing the epidermal structure.

[0058] Dermal cell arrangement: The dermal cells in the negative control group were disordered and disorganized; the cell arrangement in the positive control group was improved, but still not regular enough; the low concentration treatment had no significant effect on optimizing the dermal cell arrangement. The dermal cells in the medium and high concentration groups were more orderly and well-organized, demonstrating that the drugs prepared from the molting skin of the Anji salamander at medium and high concentrations have a better regulatory effect on the dermal cell arrangement.

[0059] Degree of inflammatory cell infiltration: The negative control group showed a large number of inflammatory cells infiltrating, indicating a significant inflammatory response; the positive control group showed a reduction in inflammatory cell infiltration, but a certain number remained; the low concentration group had a weak inhibitory effect on inflammatory cell infiltration. The medium and high concentration groups showed a significant reduction in inflammatory cell infiltration, indicating that medium and high concentrations of the experimental factor could more effectively inhibit the inflammatory response and reduce inflammatory cell infiltration. Overall, the medium and high concentrations of the drug prepared from the molted skin of the Anji salamander resulted in a more significant skin healing effect.

[0060] In Masson staining, collagen fibers are stained blue, while cytoplasm and muscle fibers are stained red. From Figure 5 The Masson staining results for each group show that in the negative control group, there was almost no obvious continuous collagen deposition in the defect area, with only scattered blue fibers distributed below the wound edge; in the positive control group, collagen deposition was slightly more abundant than in the negative group; in the low-concentration group, the collagen deposition area was wider, with the blue area clearly extending to the center of the wound, but it was relatively loose; the medium-concentration group showed the most obvious collagen deposition, with dense blue fibers appearing in both the center and edges of the wound; the high-concentration group had a similar amount of collagen deposition as the medium-concentration group. Overall, it is evident that collagen accumulation in the medium-concentration and high-concentration groups was significantly greater than that in the positive and negative control groups.

[0061] Quantitative assessment results of collagen deposition can be found in [link to relevant documentation]. Figure 6 As can be seen from the figure, the highest collagen deposition rate of the drug in the salamander molt was 68.89%. The collagen deposition rates of the low concentration, high concentration and positive control were relatively close, at 53.11%, 52.87% and 50.19% respectively, while the collagen deposition rate of the negative control group was only 35.01%.

[0062] Example 3

[0063] Efficacy evaluation of a rat model of third-degree burns

[0064] 1. Establishment of a rat model of third-degree burns: Eight-week-old female SD rats were fed according to animal health and treatment procedures (all experimental rats in this invention were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., and the entire experimental procedure complied with the "Regulations on the Management of Animal Experiments"). The rats underwent hair removal on their backs one day before modeling to observe for any allergic reactions. Rats without allergies were anesthetized by injecting 1% sodium pentobarbital at a dose of 100 μL / 10 g, according to their body weight. After disinfecting the skin on the rat's back, four symmetrical, 10 mm circular third-degree burn wounds were created along the midline of the back.

[0065] 2. Experimental grouping and treatment: The successfully modeled rats were randomly divided into 3 groups (n=6): (1) Negative control group: D-Hanks 100 μL was applied to the wound for each treatment; (2) Positive control group: Sanhuang Pearl Ointment (National Drug Approval Number Z20053321) was applied to the wound for each treatment; (3) Anji salamander molting drug group (selecting the effective concentration for skin trauma) 10ug / ml: Anji salamander molting drug was applied to the wound for each treatment.

[0066] 3. Observation indicators: (1) Recording of wound healing: Morphological changes were recorded by taking pictures with a mobile phone; (2) Histopathological analysis: Rats in each group were randomly sacrificed on the 7th and 14th days of modeling, and skin samples were taken from the modeling site, fixed with 4% paraformaldehyde, and embedded in paraffin for sectioning. Hematoxylin-eosin (H&E) staining was performed to observe the infiltration of inflammatory cells and the proliferation of fibroblasts, and Masson trichrome staining was used to assess collagen fiber deposition and tissue repair; (3) Masson staining collagen deposition assessment on the 14th day: Image J software was used to quantitatively assess the collagen deposition stained by Masson.

[0067] Result: In Figure 7 , Figure 8 In cases where wounds heal within 23 days after modeling, the medication prepared from the molted skin of the Anji salamander showed good efficacy and a significant ability to promote wound healing. Figure 9 The H&E staining results show that the negative control group is still in the early stage of burn repair and has many inflammatory cells. In the experimental group, the fibroblast layer beneath the eschar is thinner, indicating more complete repair, and the wound is almost completely healed. The positive control group has a thicker fibroblast layer and is still in the process of proliferation; the wound has not yet fully healed, and the healing effect is between the negative control and the experimental group. In Masson staining, collagen fibers are stained blue, while cytoplasm and muscle fibers are stained red. From... Figure 10The Masson staining results for each group show that the negative control had less collagen deposition and poorer repair effect; the experimental group showed clear separation of the epidermis, dermis, and muscle layer, indicating that the wound was nearing complete healing; the positive control showed more collagen deposition, and the separation of the epidermis, dermis, and muscle layer had shown a clear trend. Overall, the experimental group showed better wound healing effect than both the negative and positive control groups.

[0068] See the results of the quantitative assessment of collagen deposition. Figure 11 As can be seen from the figure, the collagen deposition rate was the highest in the experimental group on day 14, at 64.77%, followed by the positive control group at 59.84% and the negative control group at 39.32%.

[0069] Example 4

[0070] Cytotoxicity analysis validation

[0071] 1. Cell preparation: Prepare a sufficient quantity of 293T (human kidney epithelial cell line) cells.

[0072] 2. Cell plating: Once the 293T cells are growing well, plate them using the culture medium (10% FBS + DMEM + 1% PS) according to the following protocol.

[0073] Plate design: The cells were divided into experimental groups of 0.64 μg / mL, 3.2 μg / mL, 16 μg / mL, 80 μg / mL, and 400 μg / mL (n=3), a blank control group, and a negative control group: (1) Experimental group 0.64 μg / mL: 5000 cells resuspended in 100 μL of culture medium; (2) Experimental group 3.2 μg / mL: 5000 cells resuspended in 100 μL of culture medium; (3) Experimental group 16 μg / mL: 5000 cells resuspended in 100 μL of culture medium; (4) Experimental group 80 μg / mL: 5000 cells resuspended in 100 μL of culture medium; (5) Experimental group 400 μg / mL: 5000 cells resuspended in 100 μL of culture medium; (6) Blank control group: only 100 μL of culture medium was added; (7) Negative control group: 5000 cells resuspended in 100 μL of culture medium.

[0074] After adding cells and culture medium to 96-well plates using a pipette, the cells were incubated at 37°C for 24 hours. Cell growth was then observed and photographed.

[0075] 3. Cell drug administration: Using basal culture medium as the solvent, drugs prepared from the molting skin of the Anji salamander were added according to the experimental group regimens of 0.64 μg / mL, 3.2 μg / mL, 16 μg / mL, 80 μg / mL, and 400 μg / mL (n=3). The blank control group and negative control group were added with the corresponding volumes of culture medium to ensure that the culture medium volume was the same.

[0076] 4. Cell viability assay: After culturing cells for 48 hours, observe cell growth and take photographs to record the data. (See attached image) Figure 12 After photographing, discard the culture medium and add 100 μL of CCK8 assay reagent (CC8 assay reagent preparation - CC8K:DMEM = 1:9). Incubate the cell plate at 37℃ in a 5% CO2 incubator for 2-4 hours, then use a microplate reader to measure the absorbance at OD450nm, and perform numerical conversion of cell viability to assess the cytotoxicity of the drug. See the results below. Figure 13 .

[0077] Bright-field images and cell activity data show that drugs prepared from the molted skin of the Anji salamander have no cytotoxic effects at concentrations ranging from 0.64 to 400 μg / mL.

Claims

1. Application of Anji salamander molting powder in the preparation of skin wound healing drugs. The Anji salamander molting powder is obtained according to the following preparation method: a. Obtain the molted skin of the Anji salamander; b. Wash the skin five times with D-Hanks in an ice bath environment; use MagNA Lyser fully automated tissue homogenizer to fully homogenize the molted skin obtained in step a to obtain the molted skin extract of the Anji salamander. c. Transfer the *Andrias davidianus* extract obtained in step b to a centrifuge tube, filter it through a 0.22 μm filter membrane for sterilization, collect the filtrate, freeze-dry it, and obtain *Andrias davidianus* molting powder.

2. The application according to claim 1, characterized in that, The skin wound refers to a wound that extends into the dermis.

3. The application according to claim 1, characterized in that, This includes inhibiting the formation of pathological scars during the wound healing process.

4. The application according to claim 1, characterized in that, This includes inhibiting the formation of pathological scars during the wound healing process that extends deep into the dermis.

5. The application according to claim 1, characterized in that, The skin wounds are full-thickness skin defects, second- or third-degree burns, surgical wounds, or chronic ulcers.

6. A drug for promoting skin wound healing, characterized in that, It contains molted skin powder from the Anji salamander, which is prepared according to the following method: a. Obtain the molted skin of the Anji salamander; b. Wash the skin five times with D-Hanks in an ice bath environment; use MagNA Lyser fully automated tissue homogenizer to fully homogenize the molted skin obtained in step a to obtain the molted skin extract of the Anji salamander. c. Transfer the *Andrias davidianus* extract obtained in step b to a centrifuge tube, filter it through a 0.22 μm filter membrane for sterilization, collect the filtrate, freeze-dry it, and obtain *Andrias davidianus* molting powder.

7. A drug for inhibiting the formation of skin wound scars, characterized in that, It contains molted skin powder from the Anji salamander, which is prepared according to the following method: a. Obtain the molted skin of the Anji salamander; b. Wash the skin five times with D-Hanks in an ice bath environment; use MagNA Lyser fully automated tissue homogenizer to fully homogenize the molted skin obtained in step a to obtain the molted skin extract of the Anji salamander. c. Transfer the *Andrias davidianus* extract obtained in step b to a centrifuge tube, filter it through a 0.22 μm filter membrane for sterilization, collect the filtrate, freeze-dry it, and obtain *Andrias davidianus* molting powder.

8. The drug according to claim 6 or 7, characterized in that, It also includes pharmaceutically acceptable carriers.

9. The drug according to claim 6 or 7, characterized in that, The dosage forms are topical gels, ointments, sprays, dressings, or patches.

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