阅读说明：本技术 穗花杉双黄酮在制备促进伤口愈合药物中的应用 (Application of amentoflavone in preparation of medicine for promoting wound healing ) 是由 曹征宇 张凡 张壮 陈颐宸 张嫔慧 于 2021-11-26 设计创作，主要内容包括：本发明公开了穗花杉双黄酮在制备促进伤口愈合药物中的应用,穗花杉双黄酮是黄酮类小分子化合物,本发明是基于新型促进伤口愈合靶点GPR39开发一个全新激动剂。实验证明,通过激动GPR39靶点,穗花杉双黄酮具有显著的促进伤口愈合活性,可用于促进伤口愈合药物的研发。(The invention discloses application of amentoflavone in preparation of a medicine for promoting wound healing, wherein the amentoflavone is a flavonoid micromolecule compound, and a brand-new agonist is developed on the basis of a novel target spot for promoting wound healing GPR 39. Experiments prove that by exciting GPR39 target, amentoflavone has remarkable activity of promoting wound healing and can be used for research and development of medicaments for promoting wound healing.)

1. Application of amentoflavone shown in general formula (I) and pharmaceutically acceptable salt and isotope marker thereof in preparation of medicine for promoting wound healing

2. Use according to claim 1, characterized in that: the wound healing promoting drug is a GPR39 agonist.

3. Application of pharmaceutical composition containing amentoflavone in preparing medicine for promoting wound healing is provided.

Technical Field

The invention relates to a new application of a natural compound of a traditional Chinese medicine, in particular to an effect of amentoflavone in a medicine for promoting wound healing.

Background

Skin wounds are a common clinical symptom, and conventional skin wound treatment includes skin grafting and artificial substitute covering, however, the conventional methods have difficulty in satisfying the needs of clinical wound treatment due to limited donor skin sources, risk of immune rejection or infectious diseases, and high cost of artificial skin. Although some growth factors show strong effects in promoting wound healing, safety concerns have been focused due to the complex compatibility and carcinogenic potential of various growth factors involved in wound healing.

The G protein coupled receptor GPR39 is mainly expressed in skin keratinocytes in the skin system, can be activated by intercellular zinc ions, is a main regulation receptor of physiological functions related to the zinc ions in the skin, and is involved in regulating the proliferation of the keratinocytes. Recent research shows that the GPR39 gene knockout causes wound healing difficulty in mice, the GRR39 receptor is excited to promote wound healing, and GPR39 is proved to be a novel and important drug action target for regulating wound healing.

However, the discovery of GPR39 agonists with significant wound healing promoting activity is still lacking to date.

Disclosure of Invention

The purpose of the invention is as follows: the invention discloses application of amentoflavone serving as a GPR39 agonist in preparation of a medicine for promoting wound healing.

The technical scheme is as follows: the application of amentoflavone shown in general formula (I) and pharmaceutically acceptable salt and isotope marker thereof in preparing medicine for promoting wound healing,

further, the wound healing promoting drug is a GPR39 agonist.

Further, the dosage forms of the medicine for promoting wound healing comprise internal and external dosage forms.

Application of pharmaceutical composition containing amentoflavone in preparing medicine for promoting wound healing is provided.

The invention adopts a high-flux real-time fluorescence detection systemFound in a compound library of natural products of traditional Chinese medicinesGPR39 agonist amentoflavone. GPR39 is expressed primarily in keratinocytes in the skin system, regulating the proliferation and migration of keratinocytes. Using Human Immortalized Keratinocytes (HaCaT) as an in vitro model, MTT experiments and BrdU incorporation experiments were used to verify the effect of amentoflavone on cell proliferation, and the expression of GPR39 was knocked down by transfection of small interfering RNA (siRNA), and whether the effects were dependent on GPR39 was examined, indicating that amentoflavone can increase the activity of HaCaT cells dose-dependently and promote keratinocyte proliferation, and the effect was dependent on GPR 39. A mouse animal model of a full-thickness skin excision wound is established by using a 5 mm-diameter biopsy punch (Kai Industries, Tokyo, Iapan), the diameters of X, Y and Z axes of a wound are measured by using a vernier caliper (Mitir), the wound area is evaluated at each time point, the wound healing rate of a mouse is calculated, and the activity of amentoflavone for promoting wound healing is evaluated, so that the compound has stronger wound healing activity than a Recombinant Human Epidermal Growth Factor (Recombinan Human Epidermal Growth Factor, rhEGF, 2000IU/mL) at the dosage of 3 mu M.

Has the advantages that: the invention relates to a novel agonist developed based on a novel target spot for promoting wound healing GPR39, and the agonist has remarkable activity for promoting wound healing, can be used as a lead compound and is used for research and development of medicaments for promoting wound healing.

Drawings

FIG. 1 is a chemical structural formula of Amentoflavone (Amentoflavone, AF);

FIG. 2 is a graph of concentration-dependent agonism of hGPR39 of amentoflavone, wherein A is a graph of different concentrations of Amentofiavone causing GPR39 mediated cells [ Ca [²⁺]_iIncreased, panel B Amentoflavone caused GPR39 mediated cells [ Ca²⁺]_iIncreased EC₅₀Measuring;

FIG. 3 shows that amentoflavone promotes HaCaT cell proliferation via GPR 39;

FIG. 4 is a graph showing HE staining of skin wound tissue of a mouse.

Detailed Description

Example 1

The amentoflavone has a structural formula shown in figure 1 and a molecular formula of C₃₀H₁₈O₁₀Is a flavone small molecule natural product compound derived from Selaginella tamariscina (Beauv.) Spring of Selaginellaceae. Employing a high throughput real-time fluorescence detection systemThe activity of amentoflavone on heterogeneously expressed hGPR39 receptor is evaluated, and the result shows that the amentoflavone has stronger agonistic activity and EC for GPR39_s0Was 154nM as shown in FIG. 2.

MTT experiment and BrdU incorporation experiment are adopted to verify the effect of amentoflavone on keratinocyte proliferation, and whether the effects depend on GPR39 is examined by knocking down the expression of GPR39 through transfection SiRNA, the result is shown in figure 3, the amentoflavone promotes HaCaT cell proliferation through GPR39 (A: the solvent control group is 1, the numerical value of each group is the multiple of the solvent control group),^**p < 0.01, relative to the solvent control group or solvent control + perturbed small interfering RNA group;^##p is less than 0.01, relative to amentoflavone + disturbing small interfering RNA group

The MTT experiment is adopted to investigate the influence of different concentrations of AF on the cell viability after 36 hours of stimulation of HaCaT cells, and the result shows that AF can increase the HaCaT cell viability in a dose-dependent manner (FIG. 3A); with the BrdU incorporation experiment, the proportion of BrdU positive cells was significantly increased after AF (3 μ M) treatment, with a significant difference compared to the solvent control group (fig. 3B and C), indicating that AF can significantly promote keratinocyte proliferation. The promotion effect of AF on proliferation of keratinocytes is weakened after the expression of GPR39 is interfered by specific SiRNA, and the difference is very obvious compared with that of Scamble-SiRNA (FIGS. 3D and E), which indicates that the promotion effect of AF on proliferation of HaCaT cells depends on GPR 39.

A full-thickness skin excision wound mouse animal model was created using a 5 mm-diameter biopsy punch. A circular full-thickness skin excision wound of 5mm diameter was made on the back of the mouse, the X, Y, and Z axis diameters of the wound were measured using a vernier caliper, and then the wound was smeared at the wound of the mouse, and the model group mice were given 50 μ L of sterile physiological saline, the amentoflavone group: AF (3. mu.M) and AF (10. mu.M) groups were given 50. mu.L of amentoflavone formulated with sterile physiological saline; the positive drug group is administered with 50 mu L of recombinant human epidermal growth factor (2000 IU/mL); daily dosing, results of the experiment are shown in table 1.

TABLE 1 Effect of amentoflavone on mouse wound healing Rate (%)

^*，P＜0.05；^**P < 0.01, relative to model set

A5 mm-diameter biopsy punch is adopted to establish a full-thickness skin excision wound mouse animal model, the wound area is evaluated at each time point, the wound healing rate of the mouse is calculated, and the wound healing promoting activity of amentoflavone is evaluated.

The mice were sacrificed on day 8 after the model creation, the wound and the peripheral tissues were taken, fixed with 4% neutral paraformaldehyde for 48 hours, embedded in paraffin, prepared into 5 μm paraffin sections, and then HE-stained, and the pathological changes of the tissues were observed under a microscope, and the experimental results are shown in fig. 4 (S: Scab-Scab; KC: Keratinocytes-Keratinocytes; Cf: Collagen fibers-Collagen fibers; FB: fibroplasts-Fibroblasts) and table 2.

TABLE 2 Effect of amentoflavone on mouse wound epidermal thickness (μm)

^*，P＜0.05；^**P < 0.01, relative to the model set,^##p < 0.01, relative to normal group

Histopathological observation of the skin wound of the mouse at the 8 th day after the model building shows that the skin tissue of the mouse in the model group is loosely arranged, no new keratinocyte layer is seen, the whole skin structure is disordered, inflammatory cells and fibroblasts migrate to the wound, and the number of the inflammatory cells is relatively large. Compared with the two groups of amentoflavone groups and rhEGF groups, the keratinocyte layer at the wound is generated, the collagen fibers are arranged closely and orderly, a large amount of fibroblasts are visible in the wound, relatively few inflammatory cells exist, and the effect of increasing the number of the fibroblasts of the amentoflavone group is more obvious. Meanwhile, the epidermal thickness of the skin of the mice is measured, after the skin of each group of mice is wounded, the epidermal thickness is obviously thickened compared with that of the normal group of mice, and the epidermal thickness of the amentoflavone group and the rhEGF group is obviously higher than that of the model group, so that the amentoflavone and the rhEGF can promote the epidermal neogenesis, and the amentoflavone group shows more prominent wound healing promoting activity.