Application of gray fleece honeysuckle saponin A in preparation of products for promoting skin wound repair and regeneration

Abstract

The application discloses application of sibiricose A in preparation of products for promoting skin wound repair and regeneration. The application finds through experiments that sibiricose A can promote proliferation and migration of fibroblasts and vascular endothelial cells, promote lumen formation of vascular endothelial cells, and promote fibroblasts to secrete collagen I, can accelerate wound healing, promote more blood vessels and collagen to be generated, reduce secretion of pus and inflammatory substances, has the ability to promote skin wound repair and treat chronic ulcers, and provides an effective way for skin wound repair and regeneration, especially for repair and regeneration of diabetic refractory wounds.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical development for the treatment of human skin wound repair, and particularly to the application of honeysuckle saponin A in the preparation of products that promote skin wound repair and regeneration. Background Technology

[0002] The skin, the largest organ in the human body, serves not only as a protective barrier but also participates in various physiological functions. When the skin is injured, such as by cuts, abrasions, or burns, the body initiates a series of complex biological processes to repair the damage—a process known as wound healing. Wound healing is a multi-stage, continuous process, primarily consisting of four main phases: hemostasis, inflammation, proliferation, and remodeling. Throughout the healing process, multiple cells, growth factors, cytokines, and molecular pathways work synergistically to ensure effective wound closure and functional restoration. Vascular endothelial cells (HUVECs) and fibroblasts (HSFs) play crucial roles. However, this process can be influenced by various factors, including nutritional status, age, chronic diseases, infection, and wound cleanliness, all of which can delay healing or lead to poor healing. Therefore, the discovery of drugs that promote wound healing is of great value.

[0003] Natural small molecule compounds are low-molecular-weight compounds with biological activity extracted from various natural sources, including plants, microorganisms, and animals. In recent years, the application and development of small molecule natural compounds in wound healing and tissue repair have been extensively explored. Currently, widely studied natural compounds such as curcumin, resveratrol, tea polyphenols, and quercetin have been shown to have anti-inflammatory, antioxidant, and regenerative effects.

[0004] Macronthoidin A (MaA), with a molecular weight of 1237.38 and a structural formula as shown in Formula I, is an orally active saponin extracted from honeysuckle. It possesses anti-inflammatory activity and protects against liver damage induced by acetaminophen, cadmium, and carbon tetrachloride. It also significantly inhibits the swelling of soybean oil. However, to date, no information has been published regarding the use of macronthoidin A to promote skin wound healing.

[0005] Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of honeysuckle saponin A in the preparation of products that promote skin wound repair and regeneration.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Application of honeysuckle saponin A in the preparation of products that promote skin wound repair and regeneration.

[0009] The promotion of skin wound repair and regeneration includes promoting the repair and regeneration of skin cells (tissues) after trauma, as well as promoting the repair and regeneration of chronic wounds (promoting chronic wound healing).

[0010] The chronic wounds mentioned include diabetic refractory wounds, such as diabetic refractory ulcers.

[0011] The aforementioned honeysuckle saponin A (a small molecule compound) promotes the repair and regeneration of skin trauma (including chronic wounds, diabetic refractory wounds, etc.) by promoting the proliferation and migration of fibroblasts, promoting the proliferation and migration of vascular endothelial cells, promoting the formation of vascular endothelial cells lumen, and promoting the secretion of type I collagen by fibroblasts.

[0012] The fibroblasts mentioned include human fibroblasts.

[0013] The vascular endothelial cells mentioned include human vascular endothelial cells.

[0014] The blood vessels mentioned include capillaries.

[0015] The concentration of the aforementioned honeysuckle saponin A is 0.1–15 μmol / L.

[0016] The products mentioned include medical products (pharmaceutical products), skin care products, or cosmetics.

[0017] The medical products mentioned include pharmaceuticals, etc.

[0018] The product can be formulated into various dosage forms using conventional methods in the art, including solutions, lyophilized agents, emulsions, creams, gels, masks, or dressings.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] (1) Through in vitro cell experiments, namely CCK-8, EdU, colony formation and Transwell experiments, this invention found that the addition of honeysuckle saponin A can promote the proliferation and migration of HUVEC cells and HSF cells. The tube formation experiment of HUVEC cells showed that honeysuckle saponin A has the effect of promoting angiogenesis. The ELISA experiment showed that the type I collagen secretion content of HSF cells increased with the addition of honeysuckle saponin A.

[0021] (2) Through in vivo experiments, namely in chicken embryo allantoic membrane angiogenesis and transgenic zebrafish embryo vascular model, this invention found that honeysuckle saponin A showed good ability to promote angiogenesis and development; at the same time, in the back injury models of normal mice and transgenic diabetic mice, it was verified that honeysuckle saponin A can accelerate wound healing, promote more blood vessel and collagen production, reduce the secretion of pus and inflammatory substances, and has the ability and application value to promote wound repair and treat chronic ulcers.

[0022] (3) Through preliminary safety evaluation experiments, this invention found that no significant adverse reactions occurred in zebrafish larvae and mice with the addition of Lonicera japonica saponin A. Furthermore, statistical analysis of the mortality rate and dorsal deformity rate of zebrafish larvae revealed that Lonicera japonica saponin A had no significant lethal or teratogenic toxic side effects on the individual development of zebrafish larvae. In addition, in a mouse dorsal trauma model, there was no sharp decrease in mouse weight, and blood biochemistry results showed no significant changes in liver injury parameters, while the morphology of tissue sections from the heart, liver, spleen, lungs, and kidneys remained normal. These results indicate that Lonicera japonica saponin A has no significant acute toxicity, meaning that this small molecule compound can be used to prepare products that promote skin wound repair and regeneration, chronic wound healing, and the repair and regeneration of refractory diabetic wounds. Attached Figure Description

[0023] Figure 1 This is a graph showing the results of how Lonicera japonica saponin A promotes the growth and function of vascular cells and fibroblasts. Specifically: A shows the effect of different concentrations of Lonicera japonica saponin A on the proliferation activity of HSF cells, detected by EdU staining; B is a quantitative statistical graph of the effect of Lonicera japonica saponin A on the proliferation activity of HSF cells; C shows the enhancement effect of different concentrations of Lonicera japonica saponin A on the proliferation of HUVEC cells, measured and compared using a CCK-8 assay; D shows the effect of Lonicera japonica saponin A on the formation of cell colonies in HUVEC and HSF cells, evaluated by a colony formation assay; E shows the ability of Lonicera japonica saponin A to enhance the migration of HUVEC and HSF cells, detected by a Transwell assay; F shows the ability of Lonicera japonica saponin A to promote the formation of vascular endothelial cells into lumens, detected by an in vitro lumen formation assay; and G shows the ability of Lonicera japonica saponin A to promote the secretion of type I collagen by HSF cells, quantified by ELISA.

[0024] Figure 2This diagram shows the results of promoting angiogenesis and development in chicken embryos and zebrafish using Lonicera japonica saponin A. A shows the development of blood vessels in a group of 8-day-old chicken embryos treated with gradient concentrations of Lonicera japonica saponin A for 48 hours. B shows the calculation and comparison of blood vessel diameters. C shows the calculation and comparison of blood vessel coverage area. D shows the calculation and comparison of the number of blood vessel branches. E shows the development of blood vessels in newly formed zebrafish with GFP-labeled vascular endothelial cells treated with gradient concentrations of Lonicera japonica saponin A for 48 hours. F shows the statistics of blood vessel branches in the celiac venous plexus. G shows the statistics of the number of newly formed blood vessels protruding at the ends of the venous plexus. H shows the acute toxicity of gradient concentrations of Lonicera japonica saponin A. I shows the teratogenic effects of gradient concentrations of Lonicera japonica saponin A.

[0025] Figure 3 This is a diagram showing the results of how honeysuckle saponin A promotes skin wound repair in normal mice. A is a schematic diagram of the experimental procedure; B shows the modeling of a skin wound on the dorsal side of healthy C57BL / 6J mice, treated with bFGF and different concentrations of honeysuckle saponin A, with changes in wound area observed from 0 to 4 days after modeling; C is an overlay plot showing the progress of wound repair (the middle number represents the proportion of the wound area to the modeled area on day 4); D is a statistical graph of wound area changes over time; E is a statistical graph of mouse weight during the experiment; F is... Serum alanine aminotransferase (ALT) level (blood collected on the same day after mouse sacrifice, used to reflect potential liver damage); G is serum aspartate aminotransferase (AST) level (blood collected on the same day after mouse sacrifice, used to reflect potential liver damage); H is a longitudinal section of the wound, with H&E staining to distinguish tissue morphology and cell density and quantification; I is a Sirius red staining to distinguish collagen secretion and quantification (collagen is stained blue); J is an IHC staining to calculate the number and quantification of CD31-labeled endothelial cells in the wound area.

[0026] Figure 4This is a graph showing the results of promoting the repair of refractory wounds in diabetic mice using honeysuckle saponin A. A is a schematic diagram of the experimental procedure; B shows the wound area changes observed and statistically analyzed within 0–12 days after modeling with bFGF and different concentrations of honeysuckle saponin A in BKS-db mice with a diabetic background, following wound modeling; C is a stacked statistical graph of wound repair progress (the central number represents the proportion of the wound area to the modeled area on day 12); D shows the statistical changes in wound area over time; E shows the weight statistics of diabetic mice during the experiment; F shows longitudinal sections of the wound, with H&E staining to distinguish tissue morphology and cell density; G shows collagen secretion labeled by Masson staining (collagen is stained blue); H shows the number of labeled cells by labeling fibroblasts with Anti-αSMA mAb; and I shows the number of labeled vascular endothelial cells by labeling vascular endothelial cells with Anti-CD31 mAb. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0028] Example 1

[0029] 1. Honeysuckle saponin A promotes the growth and function of vascular cells and fibroblasts.

[0030] 1.1 EdU cell proliferation assay

[0031] The experiment was conducted according to the instructions for the Beyotime EdU cell proliferation assay kit, as follows:

[0032] (1) Fully digest HSF cells (ATCC) to prepare cell suspension, count the number of cells using a cell counter, and obtain a suitable cell seeding amount through preliminary experiments. 1500 cells are evenly seeded into each well of a 24-well plate.

[0033] (2) Incubate at 37℃ for 24 h to allow cells to adhere. Prepare the required drug concentrations and add them to 96-well plates. The experiment was divided into 5 groups. The experimental groups were given 2 μM, 4 μM and 8 μM of Lonicera japonica saponin A (MaA), respectively. The control group was given 500 IU of basic fibroblast growth factor (bFGF) (purchased from Shanghai Qiangyao Biotechnology Co., Ltd.). The blank control group (Blank, abbreviated as Bk) was not given any drug.

[0034] (3) When the cells reach a confluence of approximately 60%–70%, prepare a 2x EdU working solution using 10 mM EdU solution in serum-free DMEM medium (purchased from Gibco, USA). Preheat the 2x EdU solution to 37°C, then add the 2x EdU solution to an equal volume of medium containing the experimental cells to obtain a 1x EdU solution. That is, for a final EdU solution concentration of 10 μM, half of the original medium can be replaced with fresh medium containing 20 μM EdU.

[0035] (4) Place the treated cells in an incubator and continue incubation for 3 hours.

[0036] (5) After EdU labeling, remove the culture medium and add 500 μL of fixative (4% paraformaldehyde) to each well, and fix at room temperature for 15 min.

[0037] (6) Remove the fixative and wash the cells three times with 500 μL washing buffer (PBS buffer containing 3% (v / v) bovine serum albumin BSA) per well.

[0038] (7) Incubate each well with 500 μL of permeabilization buffer (PBS buffer containing 0.3% (v / v) Triton X-100) at room temperature for 10–15 min.

[0039] (8) Remove the permeation fluid, add 500 μL of washing buffer to each well, and wash the cells 3 times.

[0040] (9) Prepare the Click reaction solution according to the order and volume of the components in the kit instructions. Remove the washing buffer, add 100 μL of Click reaction solution to each well, gently shake the 24-well plate, and incubate at room temperature in the dark for 30 min.

[0041] (10) Remove the Click reaction solution, add 500 μL of washing buffer to each well, and wash the cells 3 times.

[0042] (11) Add 200 μL of diluted 1×Hochest 33342 reaction solution to each well and incubate at room temperature in the dark for 10 min.

[0043] (12) Remove the Hoches reaction solution, add 500 μL of washing buffer to each well, and wash the cells 3 times.

[0044] (13) Fluorescence was detected using a fluorescence microscope. Azide 488 was green fluorescence with a maximum excitation wavelength of 495 nm and a maximum emission wavelength of 519 nm. Hochest 33342 was blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum absorption wavelength of 460 nm. The experiment was repeated three times.

[0045] 1.2 CCK-8 cell proliferation experiment

[0046] (1) Fully digest HUVEC and HSF cells, prepare cell suspensions separately, count the number of cells using a cell counter, and determine the appropriate cell seeding amount through preliminary experiments. Evenly seed 1500 cells into each well of a 96-well plate. When seeding, be careful to mix the cell suspension from time to time to avoid cell precipitation, which would result in an uneven number of cells in each well.

[0047] (2) Incubate at 37℃ for 24 hours to allow cells to adhere. Prepare the required drug concentration and add it to each well of a 96-well plate, with the experimental groupings following the steps in 1.1 (2) above.

[0048] (3) After stimulating the cells with the drug for 48 hours, dilute the CCK-8 working solution with DMEM medium (CCK-8: medium = 1:10, v / v), mix well, remove the old medium from the test wells, and add 100ul of freshly prepared medium containing CCK-8 to each well.

[0049] (4) After culturing for an appropriate time, the OD value at 450 nm was measured using an ELISA reader (reference wavelength was 620 nm). The experiment was repeated three times.

[0050] 1.3 Cloning experiment

[0051] (1) Take HSF and HUVEC cells in the logarithmic growth phase, digest them with trypsin and gently pipette them to prepare single-cell suspensions, and count the number of cells using a Count Star cell counter.

[0052] (2) In a 6-well plate, 1000 cells are evenly distributed in each well to ensure that the cells are evenly dispersed. You can draw a cross to shake the cells evenly to ensure that there are the same number of cells in each well.

[0053] (3) Place the 6-well plate with the cells in a constant temperature incubator at 37℃ and 5% CO2 and incubate statically for several days, observing the cell growth and colony formation daily. Once the cells in the wells have grown into colony clusters of 8-16 cells each, remove the original culture medium. Add the required concentration of the drug to the wells to be tested, and place the plate back in the incubator for continued incubation, with the experimental groupings following the steps in (2) of 1.1 above.

[0054] (4) Culture for 4 to 5 days. When visible clones appear in the well plate, i.e., when the number of cells in the clone cluster reaches 50 to 80, stop the culture.

[0055] (5) Remove the culture supernatant and wash carefully twice with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15-20 min. Remove the fixative and invert the 6-well plate to air dry.

[0056] (6) Add 1 mL of 0.2% crystal violet staining solution to each well and stain for 20 min to stain the clone clusters purple. Recover the crystal violet staining solution. Slowly wash away the staining solution with running water until there is no background color interference in the well.

[0057] (7) Allow the 6-well plates to air dry naturally, take photos, count the samples, and compile statistics. The experiment was set up for three replicates.

[0058] 1.4 Transwell Experiment

[0059] (1) Change the maintenance medium (DMEM medium with a serum content of 2% (v / v), the serum was purchased from PAN, Germany) to culture the cells in the logarithmic growth phase to remove the influence of serum.

[0060] (2) HUVEC and HSF cells were digested, and cell suspensions were prepared using maintenance medium (DMEM medium with a serum content of 2% (v / v). After preliminary experiments, the appropriate cell seeding amount was determined, and the number of cells in the suspension was adjusted using a cell counter. Cells were digested with trypsin to prepare cell suspensions.

[0061] (3) Add culture medium containing 10% (v / v) FBS and the drug to the test wells (10,000 cells per well) of a 24-well plate, i.e., the lower chamber, where the experimental grouping is the same as in step (2) of 1.1 above. The culture medium in the lower chamber contains sufficient serum to provide sufficient nutrients and growth factors to induce cell migration.

[0062] (4) Place the Transwell cell in a 24-well plate, typically using a Transwell cell with a pore size of 8 micrometers.

[0063] (5) Distribute the cell suspension evenly in the upper chamber of the Transwell chamber.

[0064] (6) Continue culturing: culture in a cell culture incubator for 24 to 48 hours, the specific time depending on the results of the preliminary experiment.

[0065] (7) Use a cotton swab (loosen the tip of the swab a little so that you can wipe the corners) to gently wipe away the cells on the upper layer of the chamber.

[0066] (8) Add 500ul of 4% paraformaldehyde to each well, fix at room temperature for 20 minutes, remove the fixative, and invert the chamber to air dry.

[0067] (9) Add 0.2% crystal violet staining solution and stain for 20 minutes. Slowly wash away the staining solution until there is no background color interference.

[0068] (10) Observe the migration of cells in the chamber using a microscope and calculate the number and ratio of migrating cells. The experiment was set up to be repeated three times.

[0069] 1.5 Angiogenesis Experiment

[0070] (1) When preparing HUVEC cells, the passage number is a crucial factor. The angiogenesis effect is best when the endothelial cells are between the 2nd and 6th passages. Passages that are too early or too late will affect the experimental results.

[0071] (2) One day in advance, immerse the dispensed Matrigel (purchased from Beijing Solarbio Science & Technology Co., Ltd.) in ice and thaw slowly overnight at 4°C. All other necessary equipment, including 96-well plates, pipette tips, culture medium, and ice packs, should also be pre-cooled.

[0072] (3) Add 60 μl of matrix gel to the wells of the 96-well plate. Do not blow or agitate the matrix gel during the pipetting process to avoid generating air bubbles, and ensure that the matrix gel completely covers the bottom of the well.

[0073] (4) Place the 96-well plate with the substrate glue in an incubator at 37°C and 5% CO2 and let it stand for half an hour.

[0074] (5) During the waiting period, take HUVEC cells in the logarithmic growth phase, wash them gently with PBS, add an appropriate amount of trypsin to digest them and gently pipette them to prepare a single-cell suspension, and adjust the cell number using a Count Star cell counter.

[0075] (6) Take out the cured 96-well plate with matrix adhesive. After preliminary experiments, the appropriate cell seeding amount is 20,000 cells per well. The experimental group is given 2 μM, 4 μM and 8 μM of honeysuckle saponin A, respectively, and the control group is given 80 μL of cell suspension. Put the treated well plate back into the incubator.

[0076] (7) Blood vessels begin to form within 2 to 4 hours. The plate is removed every hour and observed under a microscope. The best time to take pictures is selected.

[0077] (8) Quantitative analysis of angiogenesis, including indicators such as the number of lumens formed and the number of vascular branches. The experiment was set up for three replicates.

[0078] 1.6 ELISA Experiment

[0079] The procedure was performed according to the instructions of the Abmart Human I Collagen Protein-Linked Immunosorbent Assay Kit. This kit uses a double-antibody sandwich method. Purified human I collagen capture antibodies are coated onto microplates to create a solid-phase antibody. Human I collagen is then added sequentially to the coated wells, followed by binding with HRP-labeled detection antibodies to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme, and then to the final yellow color under acidic conditions. The process is as follows:

[0080] (1) Adding standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard sample of different concentrations to each standard sample well.

[0081] (2) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same) and sample wells. First, add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μL of the sample to be tested (HSF cell culture supernatant) (the final sample dilution is 5 times). Add the sample to the bottom of the wells of the enzyme-labeled plate, trying not to touch the well walls, and shake to mix.

[0082] (3) Add enzyme: Add 100 μL of enzyme labeling reagent to each well, except for the blank well.

[0083] (4) Incubation: After sealing the plate with sealing film, incubate at 37°C for 60 minutes.

[0084] (5) Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0085] (6) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0086] (7) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0087] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction (at this time, the blue color will immediately turn yellow).

[0088] (9) Measurement: Zero the instrument using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution. The experiment should be repeated three times.

[0089] 2. Honeysuckle saponin A promotes the growth and function of vascular cells and fibroblasts.

[0090] 2.1 Chicken embryo allantoic membrane angiogenesis model

[0091] (1) Prepare fertilized chicken embryos (4 days after fertilization, purchased from Zhejiang Guangda Poultry Industry Co., Ltd.), check the quality by candling the eggs, and clean the eggshell surface. Place the chicken embryos horizontally in an incubator for incubation, adjust the incubator environment to 37℃ and 55% relative humidity, turn them over twice a day, and incubate until 9 days old.

[0092] (2) Remove the chicken embryo and mark the positions of the air cell and the embryo head with a light.

[0093] (3) In the clean bench, use sterile ophthalmic forceps and scissors to open a window in the air chamber, gently peel off the eggshell with the forceps, and clean up the eggshell fragments.

[0094] (4) Use a pipette to add 3-5 mL of sterile saline to moisten the shell membrane, making it easier to tear. Then aspirate the saline.

[0095] (5) Carefully remove the shell membrane with sterile forceps, being careful not to damage the vascular membrane. Observe the vascular structure and integrity, and remove any eggs that are unsuitable for the experiment.

[0096] (6) Set up control group and experimental group, and add drugs according to the set drug concentration: The experiment is divided into 5 groups. The experimental group is added with final concentrations of 0.1μM, 1μM and 10μM of Lonicera japonica saponin A, respectively. The control group is added with 500UI bFGF, and the blank control group (Blank, abbreviated as Bk) is not added with drugs.

[0097] (7) Carefully place the control group and the experimental group back into the incubator and continue incubation, but do not turn them over again.

[0098] (8) After incubation for 11 days, administer the drug for 48 hours, observe the results and take photos. The experiment was repeated three times.

[0099] 2.2 Transgenic Zebrafish Angiogenesis Experiment

[0100] (1) Preparation of test drug: The test drug is dissolved in dimethyl sulfoxide (DMSO) and physiological saline (i.e., the drug is first dissolved in DMSO, and then physiological saline is added to the dissolved drug solution for gradient dilution to ensure that the volume ratio of DMSO is less than 0.1%) to obtain the required concentration for injection.

[0101] (2) Collection and maintenance of zebrafish eggs: Adult zebrafish CZ55(y1Tg,Tg(fli1:EGFP) (CZ55(y1Tg,Tg(fli1:EGFP) was obtained from the National Zebrafish Resource Center, website: http: / / zfish.ihb.ac.cn / Article / 1401.html) were used for breeding to obtain fertilized eggs, which were then maintained in egg water at 28.5℃. The embryos were then transferred to culture dishes containing "blue" water supplemented with methylene blue (i.e., embryo culture medium in double-distilled water: NaCl 34.8g, KCl 1.6g, CaCl2·2H2O 5.8g, MgCl2·6H2O 9.78g, pH 7.2) to prevent fungal growth. The embryos were kept in the culture dishes until they reached the required developmental stage.

[0102] (3) Addition of test drugs: At the required developmental stage, the test drugs were added to the culture water of developing zebrafish larvae. The experiment was divided into 5 groups. The experimental groups were given 0.1 μM, 1 μM and 10 μM of Lonicera japonica saponin A, respectively. The control group was given 500 U of bFGF. The blank control group (Blank, abbreviated as Bk) was not given any drugs.

[0103] (4) Observation of angiogenesis: After drug administration, developing larvae were observed under a microscope to assess angiogenesis. Blood vessels were observed using a transgenic zebrafish strain expressing fluorescent proteins in their vessels. The angiogenesis response was quantified by measuring the length, density, and branching of blood vessels. The experiment was conducted in triplicate.

[0104] 3. Honeysuckle saponin A promotes skin wound repair in normal and diabetic mice.

[0105] 3.1 Mouse dorsal skin trauma model

[0106] (1) Dissolve 3% sodium pentobarbital in sterile physiological saline and anesthetize normal C57BL / 6J mice (purchased from Guangzhou Vital River Co., Ltd., 6 weeks old) and diabetic BKS-db mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd., 4 weeks old, specific strain name: BKS-Leprem2Cd479 / Gpt, strain type: Knock-out) by intraperitoneal injection, using 1 mL per kilogram of body weight.

[0107] (2) Place the mouse in a prone position and shave the fur off the surface of the mouse's back with a razor.

[0108] (3) Take an appropriate amount of hair removal cream with a cotton swab and gently apply it to the back of the mouse. Leave it for 1 minute for better hair removal results. Wipe away all hair removal cream and any remaining hair with a damp gauze.

[0109] (4) Carefully wipe the mouse skin with an alcohol swab and iodine solution.

[0110] (5) Gently lift the skin in the middle of the back, lay the mouse on its side, and use a sterile 6mm punch and ophthalmic scissors to create a full-thickness wound extending through the subcutaneous tissue, including the dartos fascia. Two symmetrical 6mm wounds are created on the back of each mouse. There are 6 mice in each group.

[0111] (6) Apply glue to one side of the silicone pad, place it on the wound to position it, and signal that the wound will not shrink too quickly.

[0112] (7) 30 μL of the drug was dripped onto the surface of two wounds on the back of the mice. In the normal C57BL / 6J mouse back skin injury model, the experimental groups were treated with 0.1 μM, 1 μM, and 10 μM of Lonicera japonica saponin A, respectively; the control group was treated with 500 UI of bFGF; and the blank control group (Blank, abbreviated as Bk) was physiological saline. The drug was dripped onto the surface of two wounds on the back of the mice (the same drug was used on both wounds). In the BKS-db diabetic mouse back skin injury model, the experimental groups were treated with 7.5 μM and 15 μM of Lonicera japonica saponin A, respectively; the control group was treated with 1000 UI of bFGF; and the blank control group (Blank, abbreviated as Bk) was physiological saline.

[0113] (8) Place the ruler under the silicone pad, measure the diameter of the wound, and take a photo.

[0114] (9) Apply sterile transparent medical dressing to create a relatively sterile and clean environment, while also securing the silicone pad.

[0115] (10) Place the mouse on a heating pad to keep it warm until it is fully awake, then return it to its cage.

[0116] (11) Observe the wound healing on the back of the mice every day, weigh them and take pictures. Administer the medication every other day for a total of 5 times.

[0117] 3.2 H&E staining experiment

[0118] (1) Fixation of specimens: Full-thickness skin samples were taken from the above-mentioned mouse skin lesions and fixed in 4% paraformaldehyde for a period of time (usually 24 to 48 hours).

[0119] (2) Dehydration and embedding: The fixed tissue specimens were dehydrated in a gradient of ethanol solutions of different concentrations (70% ethanol for 1.5 h, 85% ethanol for 1.5 h, 95% ethanol for 2 h, 100% ethanol for 30 min, 100% ethanol for 30 min, 100% ethanol for 30 min (all are volume fractions)), then immersed in a clearing agent, and finally embedded in wax.

[0120] (3) Slicing: Cut the embedded specimen into slices with a thickness of 3 to 5 micrometers.

[0121] (4) Dewaxing: Dewax the cut specimen sections with xylene, and wash with ethanol and water at various levels.

[0122] (5) Staining: Stain with hematoxylin solution for 5-20 min, rinse with tap water. Differentiate with differentiation solution for 30 s, soak in tap water for 15 min. Place in eosin solution for 2 min.

[0123] (6) Dehydration and mounting: Gradually dehydrate the stained sections until they become transparent, add neutral resin to mount them, and observe them under a microscope. The experiment was set up in triplicate.

[0124] 3.3 IHC Immunohistochemical Staining Experiment

[0125] (1) Tissue preparation: Full-thickness skin samples were taken from the above-mentioned mouse lesion sites, and fixed in paraffin using formalin or 4% paraformaldehyde. The tissue samples were then sectioned using a microtome and placed on glass slides.

[0126] (2) Dewax the paraffin sections to water and perform antigen retrieval on the sections. The antigens can be exposed by methods such as hot water hydrolysis, microwave heating or enzymatic hydrolysis.

[0127] (3) Block non-specific binding sites on the slides and block the samples with ordinary serum or other proteins to avoid false positive results.

[0128] (4) Gently shake off the blocking solution, add the prepared primary antibody (Anti-αSMA Rabbit pAb and Anti-CD31 Rabbit pAb, both purchased from Wuhan Saiweier Biotechnology Co., Ltd.) to the slice, and let it react specifically with the target molecule. Incubate overnight at 4°C.

[0129] (5) Wash away any unbound primary antibody, and add a secondary antibody (HRP-labeled) of the same species as the primary antibody (HRP-labeled goat anti-rabbit IgG, purchased from BBI Life Sciences Co., Ltd.) to specifically bind with the primary antibody. Drop the secondary antibody onto the sample and incubate for a period of time.

[0130] (6) Wash away unbound secondary antibody, develop color and stain: Add freshly prepared DAB color development solution, control the color development time under a microscope, and the positive color is brownish-yellow.

[0131] (7) Counterstain cell nuclei with hematoxylin for about 3 minutes, then rinse with tap water. Differentiate with hematoxylin differentiation solution, then rinse with tap water. Re-stain with hematoxylin blue solution, then rinse with running water.

[0132] (8) Add neutral resin and seal the slide with a coverslip to protect the slide and facilitate subsequent observation. The experiment was repeated three times.

[0133] 2. Experimental Results

[0134] 2.1 Honeysuckle saponin A promotes the growth and function of vascular cells and fibroblasts.

[0135] Vascular endothelial cells play a crucial role in tissue repair, while fibroblasts are essential for both repair and growth. Both cell types are responsive to bFGF-FGFR2-mediated growth signaling and significantly promote tissue repair and growth. In this study, we investigated the proliferative effects of Lonicera japonica saponin A on angiogenesis and cell proliferation using the human vascular endothelial cell line HUVEC and the human fibroblast line HSF. The proliferation capacity of HSF and HUVEC cells was assessed using the EdU and CCK-8 assays. The results showed that the proliferation capacity of both cell types significantly increased with increasing Lonicera japonica saponin A concentration from 0 μM to 8 μM. At a concentration of 8 μM, the proliferative effect of Lonicera japonica saponin A was comparable to that of 500 IU of exogenous bFGF (…). Figure 1 A, Figure 1 B Figure 1 C). Furthermore, a colony formation assay was performed to assess the in vitro proliferative capacity of these cells. The results showed that the number of cell clones increased with increasing concentrations of Lonicera japonica saponin A. Figure 1 D). Cell migration experiments using Transwell chambers showed that the number of HSF and HUVEC cells passing through the micropores increased with increasing concentration of Lonicera japonica saponin A, indicating that Lonicera japonica saponin A promotes cell migration. Figure 1 E). A characteristic feature of vascular endothelial cells is the formation of vascular lumens. Comparative analysis of their in vitro vascular lumen-forming properties showed that Lonicera japonica saponin A enhanced the ability of HUVEC cells to form lumens, suggesting that Lonicera japonica saponin A has the potential to promote angiogenesis. Figure 1 F). Collagen secretion is an important marker of fibroblast function. By detecting the collagen content in the supernatant of HSF cell culture, we observed a significant increase in collagen content after the addition of Lonicera japonica saponin A, indicating that Lonicera japonica saponin A induced collagen secretion in HSF cells. Figure 1 (G). Overall, the presence of Lonicera japonica saponin A enhanced the proliferation and normal physiological function of HUVECs and HSF cells. These findings suggest that Lonicera japonica saponin A has the ability to regulate cellular activities involved in tissue repair.

[0136] 2.2 Honeysuckle saponin A promotes the growth and function of vascular cells and fibroblasts.

[0137] The angiogenic properties of Lonicera japonica saponin A were evaluated in vivo using a chicken embryo angiogenesis experiment and a zebrafish embryo angiogenesis model. In the chicken embryo angiogenesis experiment, a concentration gradient of Lonicera japonica saponin A was introduced into the urosac fluid of 9-day-old chicken embryos, and the vessel diameter was measured after 48 hours. Figure 2 A). The results showed that Lonicera japonica saponin A increased the diameter of blood vessels ( Figure 2 B) Vascular area ( Figure 2 C) and the number of vascular branches ( Figure 2 D). The effect of 10 μM concentration of Lonicera japonica saponin A in promoting vascular development in chicken embryos was comparable to that of 500 IU of exogenous bFGF, indicating that it can effectively promote vascular development in chicken embryos.

[0138] To assess vascular development in zebrafish embryos, we used a zebrafish model of GFP-labeled vascular endothelial cells previously established by our team to examine and compare the development of the ventral venous plexus and dorsal artery in juvenile zebrafish treated with different concentrations of Lonicera japonica saponin A. The results showed that with increasing concentrations of Lonicera japonica saponin A, the number of prominent vascular branches in the ventral venous plexus and neovascularization gradually increased, indicating that Lonicera japonica saponin A induced an activation state of angiogenesis. Figure 2 E, Figure 2 F, Figure 2 G). Furthermore, no significant acute toxicity or teratogenic effects were observed at the highest tested concentration of 10 μM. Figure 2 H, Figure 2 I).

[0139] 2.3 Honeysuckle saponin A promotes skin wound repair in normal and diabetic mice.

[0140] This study investigated the skin repair effects of *Lonicera japonica* saponin nail on the back of normal C57BL / 6J mice and diabetic BKS-db mice (BKS-Leprem2Cd479 / Gpt). In the C57BL / 6J mouse skin wound model, wounds with a diameter of 0.8 cm were created on the back skin. Starting from the day of wound creation (Day 0), *Lonicera japonica* saponin nail was applied topically to the wound surface every two days. The wound area was photographed and measured daily until complete healing. Figure 3 A). Statistical analysis showed that all wounds healed completely within 8 days, while the groups treated with 500 IU bFGF and those treated with 10 μM Lonicera japonica saponin A healed completely within 6 days. Compared with the saline (blank) treatment group, the wound healing rate was significantly faster in the groups treated with bFGF and Lonicera japonica saponin A. In the early wound healing phase (Day 0-Day 4), the rate of wound area reduction increased with increasing Lonicera japonica saponin A concentration. Figure 3 B), the average wound area in the 10 μM Lonicera japonica saponin A group was 17.8% smaller than that in the 500 IU bFGF group. Figure 3 C). In the group treated with Lonicera japonica saponin A, the concentration of Lonicera japonica saponin A was positively correlated with the rate of wound area reduction. Figure 3 D). Furthermore, by monitoring changes in animal body weight ( Figure 3 E) and alanine aminotransferase (ALT, Figure 3 F) and aspartate aminotransferase (AST, Figure 3 Serum levels of *Lonicera japonica* saponin A were measured to assess the potential acute toxicity of *Lonicera japonica* saponin A. No significant changes in body weight or liver injury markers were observed. Subsequently, wound tissue samples were collected and sections from the wound were stained. Hematoxylin and eosin (H&E) staining of tissue sections showed that the epidermal and dermal cells in the control group were smaller and more slender. In contrast, the groups treated with 10 μM *Lonicera japonica* saponin A and 500 IU bFGF showed higher proliferative activity around the wound. Figure 3 H). Staining collagen with Sirius red showed that collagen secretion in the tissue increased with increasing concentration of Lonicera japonica saponin A. The therapeutic effect of 10 μM Lonicera japonica saponin A was comparable to that of 500 IUbFGF. Figure 3 I). Immunohistochemical (IHC) staining using CD31 monoclonal antibody showed that in the wounds treated with Lonicera japonica saponin A, the number of vascular endothelial cells increased with increasing levels of Lonicera japonica saponin A. Figure 3 These results indicate that Lonicera japonica saponin A can effectively promote wound healing in C57BL / 6J mice and plays a role in promoting angiogenesis and collagen secretion during tissue repair.

[0141] In the BKS-db diabetic mouse model, we used the same protocol as the normal skin wound model, administering different concentrations of Lonicera japonica saponin A for treatment ( Figure 4 A). On day 8, the wound area in the 15 μM Lonicera japonica saponin A group decreased by 22.3%, compared with the 1000 IU bFGF group, indicating that Lonicera japonica saponin A was more effective than bFGF in promoting early repair of diabetic refractory wounds. Figure 4 B Figure 4 C Figure 4 D). Throughout the experiment, administration of Lonicera japonica saponin A did not significantly affect the body weight of diabetic mice, indicating that no significant acute toxicity was observed in this mouse model. Figure 4 E). Histological analysis of wound skin tissue by H&E staining showed thickening of the epidermis and dermis in both the Lonicera japonica saponin A treatment group and the bFGF group. Figure 4 F). Masson staining showed a significant increase in collagen staining in the group treated with Lonicera japonica saponin nail, indicating that Lonicera japonica saponin nail promoted collagen secretion at the wound site. Figure 4G). Immunohistochemical (IHC) labeling of fibroblasts with anti-α-SMA monoclonal antibody showed that the number of fibroblasts in the epidermis and dermis of the 15 μM Lonicera japonica saponin A group and the bFGF group was greater than that in the control group. Figure 4 H). Statistical analysis also showed that the number of CD31-positive vascular endothelial cells in the 15 μM Lonicera japonica saponin A treatment group was significantly increased compared with the control group, indicating that Lonicera japonica saponin A enhanced the activity of neointima. Figure 4 I). These findings suggest that Lonicera japonica saponin A promotes fibroblast proliferation, angiogenesis, and wound repair in diabetic refractory skin lesions.

[0142] In summary, Lonicera japonica saponin A significantly improved the healing rate of normal skin wounds and diabetic refractory wounds, especially in the early repair stage of the wound.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of Lonicera japonica saponin A as the sole active ingredient in the preparation of drugs that promote skin wound repair and regeneration.

2. The application according to claim 1, characterized in that: The aforementioned promotion of skin wound repair and regeneration includes promoting the repair and regeneration of skin cells after trauma, as well as promoting the repair and regeneration of chronic wounds.

3. The application according to claim 2, characterized in that: The chronic wounds mentioned include diabetic refractory wounds.

4. The application according to claim 1, characterized in that: The aforementioned honeysuckle saponin A promotes the repair and regeneration of skin wounds by promoting the proliferation and migration of fibroblasts, the proliferation and migration of vascular endothelial cells, the formation of vascular endothelial cells lumen, and the secretion of type I collagen by fibroblasts.

5. The application according to claim 1, characterized in that: The concentration of the aforementioned honeysuckle saponin A is 0.1–15 µmol / L.

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