Use of akba in the preparation of a medicament for promoting healing of acute injury

Abstract

The application discloses application of AKBA in preparation of a medicine for promoting healing of acute injury. The application firstly proposes that AKBA is used for acute skin injury under normal immunity and normal blood sugar state, and expands the medicine range of AKBA. In a full-thickness skin excision rat wound model, AKBA promotes re-epithelialization of a wound surface by increasing expression of proliferating cell nuclear antigen (PCNA) and a proliferation marker Ki-67 (Marker of Proliferation Ki-67, Ki67), reduces inflammatory cell infiltration of skin tissue, and increases collagen fiber generation to accelerate a wound healing rate. In an in-vitro experiment, AKBA can accelerate HaCaT cell proliferation and migration, and reduce expression of inflammatory factors to promote wound healing. AKBA has a wide application prospect in promoting healing of acute injury wound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of AKBA in the preparation of drugs that promote the healing of acute injuries. Background Technology

[0002] The skin, the largest organ in the human body, is composed of multiple layers of tissue, including the epidermis, dermis, and subcutaneous tissue. It functions to regulate body temperature, provide protection, excrete waste, and sense external stimuli. Each layer plays a crucial role in maintaining normal skin function. Skin injury represents the breach of the body's first line of immune defense, and wound repair involves various regulatory mechanisms, making it a complex biological process. Wound healing generally includes the stages of hemostasis, inflammation, proliferation, and remodeling. These four stages overlap in time and influence and constrain each other. Clinically, based on the cause of wound injury, the speed of onset, the healing cycle, and the repair status, wounds are primarily classified into two categories: acute wounds and chronic wounds. Acute wounds are mostly caused by external trauma and are often short-term, severe physiological inflammation. The healing process progresses in an orderly manner through hemostasis, inflammation, proliferation, and remodeling, but they are prone to oxidative stress damage and scarring after healing. Chronic wounds, on the other hand, are often accompanied by underlying diseases such as diabetes and ischemia, existing in a microenvironment of high glucose, hypoxia, and weakened immunity, leading to persistent inflammation.

[0003] Frankincense (olibanum), a traditional Chinese medicine with a long history of clinical use, belongs to the genus *Boswellia* of the family Burseraceae. Boswellia Boswellia carterii (plant) Boswelliacarterii Birdw and related species Botanical Gardenia davidii (Birdw) Boswelliabhaw- dajiana Frankincense is the resin exuded from the bark of the Boswellia carterii tree. It has a pungent and bitter taste, is warm in nature, and enters the heart, liver, and spleen meridians. It possesses the effects of promoting blood circulation, relieving pain, reducing swelling, and promoting tissue regeneration. Frankincense is mainly composed of resin and volatile oil, and its chemical components are primarily boswellic acid (BAs) and cembranoids. BAs mainly include 3-acetyl-11-carbonyl-β-boswellic acid (AKBA), 3-hydroxy-11-carbonyl-β-boswellic acid (KBA), α-boswellic acid, and β-boswellic acid.

[0004] AKBA, as one of the main active pharmaceutical components of frankincense, exhibits a variety of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and anti-apoptotic effects. Studies have shown that hydrogels made from AKBA and basic fibroblast growth factor (bFGF) can reduce inflammation and oxidative stress, promote angiogenesis, and thus accelerate wound healing in diabetic rats. AKBA can also inhibit the levels of matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), and matrix metalloproteinase 9 (MMP-9), regulating inflammatory cells and promoting tissue repair, which may be one of the important mechanisms by which AKBA promotes the healing of chronic wounds. Furthermore, AKBA can regulate the production of inflammatory factors and reactive oxygen species (ROS) at low concentrations, protecting HaCaT cells from UVA-induced damage. Although the efficacy of AKBA in diabetic refractory wounds has been verified, its specific role in acute wound repair and the specific pathways of epidermal cell function regulation are still lacking systematic elucidation. In particular, the biological characteristics and mechanisms of action of AKBA when applied alone to wounds still need to be clarified. Summary of the Invention

[0005] Given the current state of existing technologies, which only disclose the use of AKBA in combination with other drugs to promote the healing of chronic, refractory wounds, but lack research on the mechanism of action and biological principles of AKBA alone in the repair of acute skin injuries, the main objective of this invention is to provide the application of AKBA in the preparation of drugs that promote the healing of acute injuries. Specifically, AKBA significantly accelerates the repair process of acute wounds by effectively regulating the abnormal expression of inflammatory factors in the wound microenvironment and simultaneously promoting the proliferation and migration of epidermal cells. This application provides experimental evidence and a theoretical basis for the development of single-component formulations of AKBA.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: Application of AKBA in the preparation of drugs that promote the healing of acute injuries.

[0007] The drug is a topical preparation.

[0008] The topical preparation is selected from any one of hydrogel preparations, ointments, creams, and patches.

[0009] The drug is capable of upregulating the expression of PCNA and Ki67 in wound tissue.

[0010] The drug is one that can promote the proliferation and migration of HaCaT cells.

[0011] The drug is one that can inhibit the expression of inflammatory factors.

[0012] The inflammatory factors include IL-1β, IL-6, and IL-8.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel use of AKBA alone in the treatment of acute wounds. Compared to chronic, non-healing wounds caused by diabetes, this invention is the first to propose the use of AKBA for acute skin injuries under normal immune and blood glucose conditions, thus expanding the medicinal scope of AKBA. In a rat model of full-thickness skin resection, AKBA promotes wound re-epithelialization by increasing the expression of PCNA and Ki67, reduces inflammatory cell infiltration in skin tissue, and accelerates wound healing by increasing collagen fiber production. In in vitro experiments, AKBA can accelerate HaCaT cell proliferation and migration and reduce the expression of inflammatory factors to promote wound healing. AKBA has broad application prospects in promoting the healing of acute injuries. Attached Figure Description

[0014] Figure 1 The graph shows the effect of AKBA on wound healing rate in rats; A shows the wound healing area on days 3, 5, 7, and 9; B shows a representative wound during the healing process; n=10, compared with the control group. P<0.05, P<0.01, P<0.005, P<0.001; Figure 2 Image showing HE staining results of rat wound tissue; Scale bar: 500 μm, 100 μm, n=6; Figure 3 Masson staining results of rat wound tissue; Scale bar: 500 μm, 100 μm, n=6; Figure 4 Image A shows the immunohistochemical results of PCNA in rat wound tissue; Image B shows a representative image of PCNA immunohistochemical staining in rat skin tissue; Scale bar: 500 μm, n=6, compared with the control group. P<0.05; Figure 5 Image A shows the Ki67 results of AKBA on rat wounds; A is a representative image of Ki67 immunofluorescence in rat skin tissue, and B is a quantitative image of Ki67 intensity using immunofluorescence; scale bar: 200 μm, n=6, compared with the control group. P<0.05; Figure 6Images of HE staining of normal rat wound skin with AKBA (10 μg / g); Scale bar: 500 μm, n=5; Figure 7 Image showing the effect of AKBA (0.15-2.5 μM) treatment on HaCaT cell viability after 24 h; n=4, compared with the control group. P<0.05, P<0.005, P<0.001; Figure 8 Image A shows the effect of AKBA on HaCaT cell migration over 72 hours; Image B shows a quantitative map of AKBA-induced HaCaT cell migration and a representative image of AKBA-induced HaCaT cell migration. Scale bar: 650 μm, n=3~4, compared with the control group. P<0.05; Figure 9 The figure shows the effect of AKBA on the expression of inflammatory factors in HaCaT cells induced by TNF-α; n=3, compared with the control group, ##P<0.01, ####P<0.001; compared with the model group, P<0.05, P<0.01, P<0.005. Detailed Implementation

[0015] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0016] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, all materials, reagents, etc., used in the embodiments are commercially available. Unless otherwise specified, experimental procedures are standard procedures in the art.

[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0018] The AKBA compound described in this invention is a pentacyclic triterpenoid compound, purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd., and its structure is as follows: .

[0019] Example 1 1. Experimental Methods 1.1 Hydrogel Preparation Weigh 4.0 g of carbomer 940 and slowly add it to 70 mL of water. Stir with a magnetic stirrer until dissolved, then add 2 mL of 1,2-propanediol, 4 mL of glycerol, 6.7 mL of triethanolamine, and 133.3 mg of ethylparaben. Dissolve 0.25 mg, 0.5 mg, and 1 mg of AKBA in 1 mL of 75% ethanol solution (0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively). Divide the prepared gel into four equal portions. Add different concentrations of AKBA ethanol solution to three of these portions. Add water to each system to a final volume of 100 g (for the blank matrix group, no drug is added; water is added directly to 100 g). Stir well to prepare a blank matrix and hydrogels containing 2.5 μg / g, 5 μg / g, and 10 μg / g of AKBA, respectively. Store in a sealed container at 4°C, protected from light, for later use.

[0020] As a drug that acts directly on the skin, AKBA can be formulated not only as a hydrogel, but also as an ointment, cream, patch, and other clinical topical dosage forms.

[0021] 1.2 Modeling Sixty rats were randomly divided into four groups: a blank control group, a blank matrix group, a positive control group (human epidermal growth factor gel), a low-dose group, a medium-dose group, and a high-dose group, with 10 rats in each group. After anesthetizing the rats with 1.25% tribromoethanol, the hair on their backs was shaved, and the shaved area was disinfected with 75% alcohol. A full-thickness resection wound model was then created on the back using an 8 mm diameter biopsy punch. After modeling, the rats were housed in pairs. The dressing was changed once daily; the gel was applied evenly and thinly to cover the wound, and the wound was bandaged with a bandage and breathable adhesive tape.

[0022] To investigate the potential toxicity of AKBA to normal rat skin, five rats were used. After hair removal from the back of each rat, two 3×3 cm areas of normal skin were selected and divided into a treatment group and a control group. The treatment group was treated with a hydrogel containing 10 μg / g of AKBA prepared earlier, applied topically for 9 days, while the control group received no treatment.

[0023] 1.3 Grouping and administration: The experimental administration methods and dosages are shown in Table 1.

[0024] Table 1. Experimental drug administration methods and dosages

[0025] 1.4 Sampling and Preservation On day 9 of the experiment, rats were euthanized by an overdose of 20% urethane. Skin tissue was excised 5 mm from the edge of the wound, rinsed with physiological saline, and then fixed in 10% formalin fixative.

[0026] 1.5 Preparation of pathological sections Tissue embedding: Skin tissue was removed from the fixative and dehydrated sequentially in 75%, 85%, 95%, and 100% ethanol solutions for 1 hour each. Xylene I was then immersed for 20 minutes, followed by xylene II for 30 minutes. The tissue was then soaked in paraffin I for 1 hour, paraffin II for 1.5 hours, and paraffin III for 2 hours. An appropriate amount of liquid paraffin was poured into the mold box, and the wax-soaked skin tissue was placed flat at the bottom. After the paraffin had completely solidified and cooled, the embedding frame was removed, and the wax block was trimmed.

[0027] Section preparation: Fix the trimmed wax block onto a paraffin microtome and cut it into uniform sections with a thickness of 4 μm. Gently place the wax sections into a spreader at approximately 40°C using tweezers. After the sections are flattened, retrieve them with a glass slide, place them on a 60°C slide oven for 1 hour, and then bake them in an oven for another hour.

[0028] 1.6 Hematoxylin staining Dewaxing to water: Paraffin sections were sequentially immersed in xylene I for 20 min, xylene II for 20 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then washed with distilled water.

[0029] Nuclear staining: Immerse the sections in Harris hematoxylin staining solution for 5-10 min, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water for 10 min, blue in blue solution for 5 min, and rinse with running water.

[0030] Cytoplasmic staining: Immerse the sections in eosin staining solution for 1-3 minutes. Rinse with tap water.

[0031] Dehydration and clearing mounting: The sections were sequentially immersed in 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min for dehydration and clearing. After removal and slight drying, they were mounted with neutral resin.

[0032] 1.7 Masson staining After dewaxing, place the sections in Bouin's solution and incubate overnight at room temperature or in a 37°C incubator for 2 hours for mordating. Then rinse with running water until the yellow staining on the sections disappears. Stain with azurite blue solution for 2-3 minutes, then rinse with water.

[0033] Mayer's hematoxylin staining solution was applied for 2-3 minutes, followed by rinsing with water. Differentiate with acidic ethanol solution for a few seconds, then rinse with running water for 10 minutes.

[0034] Stain with Pompadour and magenta solution for 10 minutes, then rinse briefly with distilled water.

[0035] Treat with phosphomolybdic acid solution for about 10 minutes, then proceed directly to the next step.

[0036] Stain directly with aniline blue staining solution for 5 minutes, then treat with weak acid solution for 2 minutes.

[0037] Finally, the film is dehydrated and then sealed.

[0038] 1.8 Immunohistochemical staining Antigen retrieval: After dewaxing, the tissue sections were placed in an autoclave filled with EDTA antigen retrieval buffer (pH=9.0) for antigen retrieval. The autoclave was pressured until steam began to escape, and the time was set for 3 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry out. After natural cooling, the slides were placed in phosphate buffered saline (PBS) (pH=7.4), and washed and soaked three times, 3 minutes each time.

[0039] Endogenous peroxidase inactivation: The slides were placed in an endogenous peroxidase blocking agent and incubated at room temperature in the dark for 15 min. The slides were then placed in PBS (pH=7.4) and washed and soaked three times, 3 min each time.

[0040] Blocking: Block with 3% bovine serum albumin (BSA) at room temperature for 1 h.

[0041] Primary antibody: Gently shake off the blocking solution and add the primary antibody prepared in a certain ratio. Place the slice flat in a humidified chamber and incubate overnight at 4°C.

[0042] Add reaction enhancement solution: Place the sections in PBS (pH=7.4), agitate and wash three times, 3 min each time. After slightly drying the sections, add reaction enhancement solution to the inside of the tube to cover the tissue, and incubate at 37°C for 20 min.

[0043] Enhancing polymer: The tissue sections were placed in PBS (pH=7.4) and washed and soaked three times, 3 min each time. After slightly drying the sections, the enhancing polymer was dropped into the circle to cover the tissue, and incubated at 37°C for 20 min.

[0044] 3,3'-Diaminobenzidine (DAB) staining: The slides were placed in PBS (pH=7.4) and agitated and washed three times, 3 min each time. After slightly drying the slides, freshly prepared DAB staining solution was added to the circle. The staining time was controlled under a microscope. A positive result was brownish-yellow. The staining was stopped by rinsing the slides with tap water.

[0045] Counterstaining: Counterstain with hematoxylin for about 5 seconds, then rinse with tap water.

[0046] Finally, the film is dehydrated, cleared, and sealed. 1.9 Immunofluorescence staining Antigen retrieval: After dewaxing, the tissue sections were placed in an autoclave filled with EDTA antigen retrieval buffer (pH=9.0) for antigen retrieval. The timer was started 3 minutes after the pressure in the autoclave began to rise and steam was released. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be dried out. After natural cooling, the slides were placed in PBS (pH=7.4) and washed and soaked 3 times, 3 minutes each time.

[0047] Blocking: After slightly drying the sections, draw a circle around the tissue with a histochemical pen (to prevent antibody from flowing away), add 1% BSA evenly to the histochemical circle, and block at room temperature for 1 h.

[0048] Primary antibody: Gently shake off the blocking solution, add the prepared primary antibody in a certain proportion to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight.

[0049] Secondary antibody: Place the sections in PBS (pH=7.4), agitate and wash three times, 3 min each time. After slightly drying the sections, add the fluorescent secondary antibody corresponding to the species of the primary antibody to the inner circle in the dark, cover the tissue, and incubate at room temperature in the dark for 1 h.

[0050] DAPI counterstaining of cell nuclei: The sections were placed in PBS (pH=7.4) and washed and soaked three times, 10 min each time. After the sections were slightly dried, DAPI nuclear staining solution was added to the circle and stained at room temperature for 5 min.

[0051] Mounting: Place the slides in PBS (pH=7.4), agitate and wash three times, 5 min each time. Observe the target antibody and cell nuclear fluorescence under a fluorescence microscope, and mount with anti-fluorescence quenching mounting medium.

[0052] 1.10 Statistical Analysis Statistical analysis and graphing were performed using GraphPad Prism 9 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0053] 2. Experimental Results 2.1 Effect of AKBA on wound healing area in rats Photos of the wound were taken and statistically analyzed on days 0, 3, 5, 7, and 9. Figure 1 The statistical results of the percentage of wound area in each group of rats in Table 2 showed that, compared with the control group, the skin healing speed was faster in the positive drug group and the drug-treated group, while there was no significant difference in the matrix group. This indicates that AKBA has the effect of promoting the healing of skin defects.

[0054] Table 2 Effects of AKBA on wound healing in rats

[0055] Note: Compared with the control group, P<0.05, P<0.01, P<0.001, P<0.0001.

[0056] 2.2 Analysis of histopathological results of rat skin tissue Figure 2 HE results showed that, compared with the control group and the matrix group, the positive drug group and the drug-treated group had reduced inflammatory cell infiltration in the skin tissue, accelerated reepithelialization process and significantly increased collagen fiber density. Figure 3 Masson staining showed that, compared with the control group and the matrix group, the positive drug group and the AKBA-treated group had increased collagen fibers in their skin tissue and regular tissue arrangement.

[0057] Immunohistochemical results as follows Figure 4 As shown, compared with the control group and the matrix group, the high-dose AKBA group (10 μg / g) exhibited better healing speed and quality, increased the expression of PCNA in rat skin tissue, and significantly enhanced the cell proliferation activity of wound tissue.

[0058] Immunofluorescence assay results as follows Figure 5 As shown, compared with the control group and the matrix group, the expression level of Ki67 in the wound tissue of each group was increased to varying degrees. Among them, the high-dose AKBA group (10 μg / g) significantly increased the expression of Ki67 in rat skin tissue and promoted cell proliferation in wound tissue for wound repair.

[0059] This experiment investigated the potential toxicity of AKBA to normal skin by applying AKBA hydrogel topically. The results are as follows: Figure 6 As shown, from an appearance perspective, no adverse reactions such as erythema, edema, and desquamation occurred in the skin after AKBA administration. HE results showed that, compared with the normal group, the skin in the AKBA-treated group did not show inflammatory cell infiltration, collagen fibers were arranged in an orderly manner, and the skin hair follicle structure remained intact.

[0060] Example 2 1. Experimental Methods 1.1 Cell Counting Kit-8 (CCK-8) Viability Assay Harvested HaCaT cells in good growth condition were counted using a cell counter and the cell density was adjusted to 1×10⁵ cells / mL. 100 μL of cell suspension was added to each well of a 96-well plate and cultured for 24 h. The supernatant was discarded, and complete culture medium solutions containing different concentrations of AKBA (0, 0.15, 0.31, 0.63, 1.25, 2.5 μM) were added. The plates were then incubated for another 24 h. After incubation, the culture medium was discarded, and CCK-8 working solution was prepared according to the manufacturer's instructions and added to the 96-well plates. The plates were incubated at 37°C in the dark for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated using the formula.

[0061] 1.2 Scratch Test HaCaT cells in good growth condition were seeded into 12-well plates. When the cell confluence reached 90%, three scratches were made perpendicular to the bottom of each well using a 200 μL sterile pipette tip. The cells were then rinsed with PBS to remove any remaining cell debris and culture medium. Culture medium containing different concentrations of AKBA was then added, and the cells were incubated. Images of the scratches in each region were taken using a fluorescence microscope at different time points, and the images were statistically analyzed using Image-Pro Plus 6.0 software.

[0062] 1.3 Detection of IL-6, IL-1β, and IL-8 expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) Cell modeling and drug treatment: HaCaT cells in good growth condition were seeded in 6-well plates. When the cells reached approximately 60% confluence, the old culture medium was discarded. The experimental group was treated with complete culture medium containing tumor necrosis factor-alpha (TNF-α) (10 ng / mL) and different concentrations of AKBA (0.31, 0.63, 1.25 μM). The modeling group was treated with the same volume of TNF-α (10 ng / mL), and the control group was treated with the same volume of dimethyl sulfoxide (DMSO) solution. After culturing for another 24 h, the cells were collected in 1.5 mL Eppendorf tubes and stored at -20°C.

[0063] RNA extraction: Add 1 mL of Trizol to each centrifuge tube, vortex to mix, incubate on ice for 5 min, then add 200 μL of RNA Extraction Agent, and repeatedly pipette until no obvious precipitate appears in the lysis buffer. Incubate on ice for 10 min, then centrifuge at 4°C / 12000 rpm for 15 min. Transfer 400 μL of supernatant to a new enzyme-free Eppendorf tube, add 400 μL of isopropanol, invert to mix, incubate for 10 min, then centrifuge at 4°C / 10000 rpm for 10 min. Discard the supernatant, add 1 mL of 75% ethanol, vortex vigorously, centrifuge at 4°C / 8000 rpm for 5 min, and discard the supernatant. Add another 1 mL of 75% ethanol and repeat the washing steps. Discard the supernatant, open the tube and dry for 15 min, then add 20 μL of RNA lysis buffer to dissolve the RNA. Determine the RNA concentration using a micro-ultraviolet spectrophotometer and store at -80°C.

[0064] Reverse transcription: Add reverse transcription reagents according to the table below and perform reverse transcription in a metal bath.

[0065] Table 3. Reverse Transcription System Preparation Table

[0066] Reverse transcription conditions: 37°C for 15 min, 85°C for 5 s, 4°C for any time, and store at -20°C.

[0067] Amplification Amplification is performed by adding cDNA and corresponding reagents according to the amplification system in the table below.

[0068] Table 4. Preparation of Amplification System

[0069] The primers were synthesized by Beijing Dingguo Co., Ltd., and designed by NCBI. The primer sequences are shown in the table below: Table 5 Gene Primer Sequences

[0070] Reaction conditions: Pre-denaturation: 95°C, 30 s; Denaturation: 95°C, 10 s; Annealing / Extension: 60°C, 30 s; Denaturation, annealing, and extension were performed for 40 cycles.

[0071] Results Analysis Gene expression was detected in each sample using a PCR instrument to obtain the Ct value of the sample. The relative ΔCt of the sample was calculated, i.e., ΔCt = Ct target gene - Ct internal reference gene. After comparison with the control group, the 2 - ΔCt value was used for statistical analysis and plotting.

[0072] 1.5 Statistical Analysis Statistical analysis and graphing were performed using GraphPad Prism 9 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0073] 2. Experimental Results 2.1 Effects of AKBA on HaCaT cell proliferation The effects of different concentrations of AKBA on the proliferation of HaCaT cells were screened using the CCK-8 assay. Figure 7 As shown, AKBA can promote HaCaT cell proliferation at low concentrations (0.31–1.25 μM).

[0074] 2.2 Effects of AKBA on HaCaT cell migration HaCaT cell migration is a crucial part of wound healing, involving reepithelialization. Therefore, this study used a scratch assay to verify the effect of AKBA on HaCaT cell migration. Results are as follows: Figure 8 As shown, the migration rate of the 0.63 μM drug group (58.57%) was significantly higher than that of the control group (45.61%), and the experimental results were statistically significant. This demonstrates that AKBA can promote HaCaT cell migration.

[0075] 2.3 Effects of AKBA on TNF-α-induced expression of inflammatory factors in HaCaT cells To further investigate the effect of AKBA on wound inflammation relief, RT-qPCR was used to detect the influence of AKBA on the expression of inflammatory factors in TNF-α-induced HaCaT cells. Figure 9 The results showed that, compared with the normal group, TNF-α treatment significantly increased the mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-8. AKBA significantly reduced the mRNA expression levels of inflammatory cytokines at 0.63 μM and 1.25 μM. These results indicate that AKBA can significantly reduce the levels of inflammatory cytokines in HaCaT cells, thereby promoting wound healing.

[0076] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. Application of AKBA in the preparation of drugs that promote the healing of acute injuries.

2. The application of AKBA according to claim 1 in the preparation of a drug for promoting acute injury healing, characterized in that, The drug is a topical preparation.

3. The application of AKBA according to claim 2 in the preparation of drugs that promote acute injury healing, characterized in that, The topical preparation is selected from any one of hydrogel preparations, ointments, creams, and patches.

4. The application of AKBA according to claim 1 in the preparation of drugs that promote acute injury healing, characterized in that, The drug is capable of upregulating the expression of PCNA and Ki67 in wound tissue.

5. The application of AKBA according to claim 1 in the preparation of a drug for promoting acute injury healing, characterized in that, The drug is one that can promote the proliferation and migration of HaCaT cells.

6. The application of AKBA according to claim 1 in the preparation of a drug for promoting acute injury healing, characterized in that, The drug is one that can inhibit the expression of inflammatory factors.

7. The use of AKBA according to claim 6 in the preparation of a drug for promoting acute injury healing, characterized in that, The inflammatory factors include IL-1β, IL-6, and IL-8.

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