Use of small molecule polypeptide elabela in diabetic ulcer wound healing

Abstract

Provided is a use of a small molecule polypeptide Elabela in diabetic ulcer wound healing. Specifically disclosed is a use of Elabela in preparation of a drug for promoting diabetic ulcer wound healing. In vivo experiments (a full-thickness skin defect model established using mice) and in-vitro experiments prove the positive effect of Elabela in diabetic wound healing. Specifically, Elabela significantly promotes wound healing of diabetic mice, reduces expression of inflammatory factors (IL-6, TNF-α) and a DNA damage product 8-OHdG, reduces inflammatory exudation, can further improve the migration capability of endothelial cells and promote the tubule formation of endothelial cells, and provides a candidate drug for treatment of diabetic ulcer wounds.

Description

Application of small molecule peptide Elabela in wound healing of diabetic ulcer Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to application of the small molecule polypeptide Elabela in healing of diabetic ulcer wounds. Background Art

[0002] According to WHO data, the prevalence of diabetes has increased fourfold in the past 40 years and has become one of the most common chronic diseases. 15% of diabetic patients worldwide will experience foot complications, which are a major cause of amputation and even death. One of the key reasons why diabetic foot wounds are difficult to heal is that persistent inflammatory microenvironment damages vascular endothelial cells, resulting in a reduced ability of endothelial cells to restore and regenerate microvessels through the angiogenesis process. Endothelial dysfunction occurs before diabetes-related microvascular and macrovascular complications. Elabela (ELA) is the second largest endogenous ligand discovered in recent years and is presumed to be the angiotensin homologous receptor APJ. It has been found to have a strong pro-angiogenic effect and can promote endothelial development and angiogenesis through multiple pathways.

[0003] Summary of the Invention

[0004] The first aspect of the present invention aims to provide the use of Elabela in the preparation of a medicament for promoting the healing of diabetic ulcer wounds.

[0005] The second aspect of the present invention aims to provide the use of Elabela in improving cell migration ability and / or preparing a drug for improving cell migration ability.

[0006] The third aspect of the present invention aims to provide the use of Elabela in improving the angiogenesis ability of cells and / or preparing a drug for improving the angiogenesis ability of cells.

[0007] The fourth aspect of the present invention aims to provide a method for improving cell migration ability.

[0008] The fifth aspect of the present invention aims to provide a method for improving the angiogenesis ability of cells.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention is to provide the use of Elabela in the preparation of a medicament for promoting the healing of diabetic ulcer wounds.

[0011] Preferably, the drug is a drug that reduces the expression level of inflammatory factors.

[0012] Preferably, the inflammatory factors include IL-6 and / or TNF-α.

[0013] Preferably, the drug is a drug that improves cell migration ability; further, the cells are endothelial cells.

[0014] Preferably, the drug is a drug that improves the angiogenesis ability of cells.

[0015] Preferably, the drug is a drug that improves the tubule-forming ability of endothelial cells.

[0016] Preferably, the drug is a drug that reduces the expression level of the DNA damage product 8-OHdG.

[0017] Preferably, the medicine includes tablets, capsules, granules, pills, emulsions, liquids, patches, ointments and injections.

[0018] Preferably, the effective dose of Elabela in the drug is 3-10 μg / g; further 5-8 μg / g.

[0019] The second aspect of the present invention is to provide the use of Elabela in improving cell migration ability and / or preparing a drug for improving cell migration ability.

[0020] Preferably, the cells comprise endothelial cells.

[0021] Preferably, the concentration of Elabela is 3-10 μmol / L; further 5-8 μmol / L.

[0022] The third aspect of the present invention is to provide the use of Elabela in improving the angiogenesis ability of cells and / or preparing a drug for improving the angiogenesis ability of cells.

[0023] Preferably, the cells comprise endothelial cells.

[0024] Preferably, the effective dose of Elabela is 3-10 μmol / L; further 5-8 μmol / L.

[0025] A fourth aspect of the present invention is to provide a method for improving cell migration ability, comprising contacting cells with Elabela, wherein the cells are endothelial cells.

[0026] Preferably, the effective dose of Elabela is 3-10 μmol / L; further 5-8 μmol / L.

[0027] Preferably, the cells are human umbilical vein endothelial cells.

[0028] A fifth aspect of the present invention is to provide a method for improving the angiogenesis ability of cells, comprising contacting cells with Elabela, wherein the cells are endothelial cells.

[0029] Preferably, the effective dose of Elabela is 3-10 μmol / L; further 5-8 μmol / L.

[0030] Preferably, the cells are human umbilical vein endothelial cells.

[0031] The beneficial effects of the present invention are:

[0032] The present invention demonstrates the positive role of Elabela in diabetic wound healing through in vivo experiments (a full-thickness skin defect model constructed in mice) and in vitro experiments. Specifically, Elabela significantly promotes wound healing in diabetic mice, reduces the expression of inflammatory factors (IL-6, TNF-α) and the DNA damage product 8-OHdG, and reduces inflammatory exudate. At the same time, it can also enhance the migration ability of endothelial cells and promote the tubule formation of endothelial cells, providing a candidate drug for the treatment of diabetic ulcer wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic diagram of the animal modeling process.

[0034] Figures 2A-2E show the results of a validation experiment using Elabela for diabetic ulcer wound healing; Figure 2A shows localized wound images and wound healing area analysis of diabetic mice (db / db) and control mice (db / m) (n=5); Figure 2B shows the expression levels of inflammatory factors (IL-6, TNF-α, and 8-OHdG) in mouse serum; Figure 2C shows photographs and area analysis of the same site in the scratch healing experiment at 0 h and 24 h; Figure 2D shows representative images of crystal violet-stained chamber cells and cell number analysis in a Transwell experiment; Figure 2E shows an in vitro angiogenesis assay of endothelial cells and analysis of the number of tubes formed. In each figure, * represents p<0.05, ** represents p<0.01, *** represents p<0.005, and **** represents p<0.001. DETAILED DESCRIPTION

[0035] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0036] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0037] The experimental data presented in this paper are the mean ± SD obtained from three independent replicates. Data were analyzed using GraphPad Prism 9.0. Normal distribution was confirmed by normality test, and Student's t test was used for comparison of data between two groups. Comparisons of more than two groups were examined by Tukey's test and one-way ANOVA analysis of variance. A p value < 0.05 was considered statistically significant.

[0038] The Elabela used in the present invention was purchased from Jier Biochemical Company (Shanghai), #585575.

[0039] Example 1 In vivo experiment on the effect of Elabela on wound healing in diabetic mice

[0040] First, a full-thickness skin defect model was constructed in diabetic mice: a total of 5 db / m mice (9 weeks, 20-30 g, male) and 10 db / db mice (9 weeks, 45-55 g, male) were purchased from Shenzhen Top Biotechnology Co., Ltd. (Shenzhen, China). Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals. This protocol has been approved by the Ethics Committee of the Eighth Affiliated Hospital of Sun Yat-sen University (No. 2022-051-01). After one week of adaptive feeding, the mice were used to construct a full-thickness skin defect model. The mice were anesthetized with 1% pentobarbital, the hair on the skin surface was removed, and a 1.0 cm circular incision was made in the center of the mouse's back. The wound was bandaged with sterile gauze, and each mouse was housed individually.

[0041] Intervention: db / db mice were randomly assigned to a modeling control group (db / db) or a modeling drug-treated group (db / db+ELA), with five mice in each group. db / m mice served as the control group. As shown in the animal modeling process diagram (Figure 1), the modeling drug-treated group (db / db+ELA) received subcutaneous injections of 5 μg / g / day at multiple sites around the wound edge on days 1, 3, 5, 7, 9, 11, and 13. The control group (db / m) and the modeling control (db / db) group received the same dose of PBS solution. At designated time points, wounds were photographed to document healing progress, mouse weights were recorded using an electronic balance, and blood glucose levels were recorded using a glucometer. On day 21, full-thickness skin tissue (0.1 cm) from the wound edge was excised and stored at -80°C.

[0042] As shown in Figure 2A, the wounds of the control db / m non-diabetic mice were almost completely healed on the 14th day, while the wounds of the db / db group mice had more exudate around the wounds and there were still obvious unhealed areas on the 21st day. The overall speed of wound healing in the modeling and drug-added group was significantly faster than that in the db / db group. As shown in the line graph, on the 21st day, the wound healing rate basically reached 100%. The above experiments show that Elabela can promote wound healing in diabetic mice.

[0043] The expression of inflammatory factors (IL-6, TNF-α) and DNA damage product 8-OHdG in the serum of mice on the 21st day of administration was further detected by ELISA. As can be seen from Figure 2B, the levels of inflammatory factors and DNA damage product 8-OHdG in the control group were the lowest, while compared with diabetic mice, the levels of inflammatory factors and 8-OHdG in the modeling and drug-addition group were significantly reduced, with significant differences (p<0.05).

[0044] Example 2 Wound healing and migration experiments of human umbilical vein endothelial cells

[0045] Cell Pretreatment: Primary human umbilical vein endothelial cells (HUVECs) were purchased from iCell Bioscience (Shanghai, China) and cultured in ECM medium containing 5.5 mmol / L glucose (ScienCell, USA) in a 5% CO2, 37°C incubator. Cells were passaged from the second to seventh passages and used for experiments. HUVECs were randomly divided into three groups: a blank group (BL), a high glucose plus ELA group (HG+ELA), and a high glucose group (HG). Cells in the blank group (BL) were grown in ECM medium containing 5.5 mmol / L glucose, while cells in the HG and ELA groups were grown in ECM medium containing 30 mmol / L glucose (Sigma, Japan). Cells were treated with Elabela (final concentration of 5 μM) for 48 hours and then used.

[0046] Wound healing assay: 1×10 6 Cells were pretreated with different methods. When the cell density reached 90%, wounds were created using a 10 μL pipette tip. Floating cells were removed with PBS and then grown in 1% serum medium. Wounds were recorded using an inverted microscope at 0 and 24 hours. Wound area was analyzed using Image J. Wound closure rate was calculated as: Wound closure (%) = [(Area0 h - Area24 h) / Area0 h] × 100%.

[0047] The results are shown in Figure 2C. The 24-hour high glucose (HG) group had an endothelial cell scratch healing rate of approximately 17.5%, which was much lower than the 22.75% in the high glucose-dosed group, indicating that high glucose significantly inhibited the scratch healing ability of endothelial cells and could be partially reversed by Elabela.

[0048] Cell migration experiment: A transwell chamber with a 24-well plate (Corning, USA) with a pore size of 8.0 μm was used. 5×10 5Cells suspended in 1% serum medium were placed in the upper chamber, and the supernatant was removed after 24 hours. The chamber was washed and fixed with 4% paraformaldehyde at room temperature, followed by staining with 0.1% crystal violet solution for 20 minutes. The cells in the upper chamber were then gently wiped with a cotton swab. Three randomly selected, non-overlapping areas were imaged. Cells in the lower chamber were counted for statistical analysis using Image J.

[0049] The results are shown in Figure 2D. Compared with the control group, the number of endothelial cells in the lower chamber was significantly reduced after cells were cultured in high glucose (HG) for 48 h, while the number of cells in the HG+ELA group was significantly greater than that in the high glucose group, suggesting that the migration ability of endothelial cells was partially reversed by Elabela.

[0050] Angiogenesis Assay: Matrigel (Corning, USA) was used to assess HUVEC angiogenesis. Experimental reagents and consumables were pre-chilled at 4°C. Matrigel was diluted with PBS (Matrigel:PBS = 1:1), and 50 μL of the diluted mixture was added to each well of a 96-well plate. After incubation for 1 hour, 2 × 10 4 The endothelial cells were imaged using an inverted microscope (Olympus, Japan) after 6 hours of incubation. The total number of tubes was quantified using ImageJ, as well as the length of the tubes. Data are from three independent replicates. Comparisons of multiple groups were performed using Tukey's test and one-way ANOVA analysis of variance. p values ​​< 0.05 were considered statistically significant.

[0051] The results are shown in Figure 2E. At the 6th hour, the tubules were fixed and photographed. The results showed that the endothelial cells in the control group could form extensive and dense large and small tubules, while there were almost no large tubules in the HG group and small tubules were rare. The exogenous administration of Elabela restored the tubule-forming ability of some endothelial cells, and the statistical counts were statistically significant (p < 0.05).

[0052] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of Elabela in the preparation of drugs for promoting healing of diabetic ulcer wounds.

2. The use according to claim 1, characterized in that: The drug is a drug that reduces the expression level of inflammatory factors.

3. The use according to claim 2, characterized in that: The inflammatory factors include IL-6 and / or TNF-α.

4. The use according to claim 1, characterized in that: The drug is a drug for improving cell migration ability.

5. The use according to claim 1, characterized in that: The drug is a drug for improving the angiogenesis ability of cells.

6. Use of Elabela in improving cell migration ability and / or preparing drugs for improving cell migration ability.

7. Use of Elabela in improving the angiogenesis ability of cells and / or preparing drugs for improving the angiogenesis ability of cells.

8. The use according to claim 6 or 7, characterized in that: The cells include endothelial cells.

9. A method for improving cell migration ability, characterized in that: Cells are contacted with Elabela, and the cells are endothelial cells.

10. A method for improving the angiogenesis ability of cells, characterized in that: Cells are contacted with Elabela, and the cells are endothelial cells.

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