阅读说明：本技术 吴茱萸碱在制备预防和/或治疗瘢痕的药物中的应用 (Application of evodiamine in preparation of medicine for preventing and/or treating scars ) 是由 郑勇军 舒付婷 武善超 盛春泉 张伟 陆剑瑜 王雨翔 肖仕初 夏照帆 于 2021-10-13 设计创作，主要内容包括：本发明提供了一种吴茱萸碱在制备预防和/或治疗瘢痕的药物中的应用,属于医药生物技术领域。吴茱萸碱通过抑制瘢痕肌成纤维细胞细胞外基质的分泌、抑制瘢痕成纤维细胞的增殖、促进瘢痕成纤维细胞的凋亡以及抑制瘢痕成纤维细胞的细胞骨架的活性来预防和/或治疗瘢痕。(The invention provides application of evodiamine in preparation of a medicine for preventing and/or treating scars, and belongs to the technical field of medical biology. The evodiamine can be used for preventing and/or treating scars by inhibiting secretion of extracellular matrix of scar muscle fibroblasts, inhibiting proliferation of scar fibroblasts, promoting apoptosis of the scar fibroblasts and inhibiting activity of cytoskeleton of the scar fibroblasts.)

1. Application of evodiamine in preparing medicine for preventing and/or treating scar is provided.

2. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the scar comprises immature scar, mature scar, atrophic scar, hypertrophic scar, keloid scar and systemic scleroderma caused by myofibroblast activation.

3. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

the medicament is a medicament for preventing and/or treating scar by inhibiting secretion of scar myofibroblast extracellular matrix.

4. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

the medicament is a medicament for preventing and/or treating scars by inhibiting proliferation and migration of scar fibroblasts.

5. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the medicament is a medicament for preventing and/or treating scar by promoting apoptosis of scar fibroblast.

6. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the medicament is a medicament for preventing and/or treating scar by inhibiting the activity of cytoskeleton of scar fibroblast.

7. The use of evodiamine, according to claim 1, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the scar is a mammalian scar.

8. The use of evodiamine, according to claim 7, in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the mammal is a rabbit.

9. The use of evodiamine according to claim 7 or 8 in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the application concentration of the evodiamine is 10-100 μ M.

10. The use of evodiamine according to claim 7 or 8 in the preparation of a medicament for the prevention and/or treatment of scarring, characterised in that:

wherein the administration mode of the fructus evodiae is intrascar local injection.

Technical Field

The invention relates to the technical field of medical biology, in particular to application of evodiamine in preparation of a medicine for preventing and/or treating scars.

Background

Scars cause the change of the normal structure of the skin, the appearance is affected, and the pruritus and pain caused by the scar formation seriously affect the life quality of patients. Moreover, severe scarring causes joint contractures, limiting joint movement and resulting in deformity. There are a lot of anti-scar drugs in clinic, such as hormone drugs like triamcinolone acetonide which is injected locally, asiaticoside cream ointment, compound heparin sodium-urea-cystine gel, silicone ointment, etc., but all have the defects of poor curative effect, long treatment time, high price, great side effect, etc.

The mechanism of scarring is not yet fully understood. Normal fibroblast cells in the scar are transformed into myofibroblast cells, and then proliferate in a large amount, resist apoptosis, and secrete extracellular matrices such as collagen, which is considered as a main mechanism of scar formation. Meanwhile, myofibroblasts over-express smooth muscle actin (alpha-SMA), which can cause scar contracture. The cytoskeleton is a protein fiber net rack system in eukaryotic cells, comprises microtubules, microfilaments and intermediate fibers, and plays an important role in maintaining the cell morphology, bearing external force, keeping the normal proliferation, migration and contraction of cells and the like. Theoretically, inhibition of the cytoskeleton of scar fibroblasts could improve scar formation.

The evodiamine is one of the most important alkaloid components of the traditional Chinese herbal medicine fructus evodiae, is known for its versatility, is used for treating diseases such as vomit, headache, diarrhea, abdominal pain and the like, is easy to extract and has high clinical treatment safety. Meanwhile, the evodiamine plays an important role in resisting tumors and can inhibit the cytoskeleton formation of tumor cells, thereby inhibiting the proliferation of the tumor cells and promoting the apoptosis. However, no report of the application of evodiamine in scar treatment is found at present.

Disclosure of Invention

The invention is carried out to solve the problems and aims to provide the application of evodiamine in preparing the medicament for preventing and/or treating scars.

The invention provides application of evodiamine in preparation of a medicament for preventing and/or treating scars.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein the scar comprises immature scar, mature scar, atrophic scar, hyperplastic scar, keloid and systemic sclerosis caused by myofibroblast activation.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: the medicament is a medicament for preventing and/or treating scars by inhibiting secretion of extracellular matrix of scar muscle fibroblasts.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: the medicament is a medicament for preventing and/or treating scars by inhibiting proliferation and migration of scar fibroblasts.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: the medicament is a medicament for preventing and/or treating scars by promoting apoptosis of scar fibroblasts.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein the medicament is a medicament for preventing and/or treating scar by inhibiting the activity of cytoskeleton of scar fibroblast.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein the scar is mammalian scar.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein the mammal is rabbit.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein the concentration of evodiamine is 10-100 μ M.

In the application of the evodiamine provided by the invention in the preparation of the medicament for preventing and/or treating the scar, the evodiamine also has the following characteristics: wherein, the administration mode of the fructus evodiae is local injection in scars.

Action and Effect of the invention

Evodiamine is widely researched, corresponding pharmacology, pharmacokinetics, toxicology and the like are relatively perfect, and the local application of the evodiamine in scar resistance is safe, effective and low in price.

In addition, the evodiamine can be used for preventing and/or treating scars by inhibiting the secretion of extracellular matrix of scar muscle fibroblasts, inhibiting the proliferation of scar fibroblasts, promoting the apoptosis of the scar fibroblasts and inhibiting the activity of cytoskeleton of the scar fibroblasts.

In addition, triamcinolone acetonide, asiaticoside cream, compound heparin sodium urocystine, silicone gel, etc. are the anti-scar drugs on the market at present, and the scar hyperplasia is relieved mainly by causing local hypoxia of the scar and inhibiting local inflammatory reaction. The evodiamine in the invention has different action mechanisms with the medicines, so the evodiamine can be used with the medicines synergistically to achieve better scar prevention and/or treatment effects.

Drawings

FIG. 1 is a general view of evodiamine induced apoptosis of scar fibroblasts in a first embodiment of the present invention;

FIG. 2 is a flow chart of the results of evodiamine induced apoptosis of scar fibroblasts in accordance with the first embodiment of the present invention;

FIG. 3 is a graph showing the results of evodiamine inhibiting proliferation of scar fibroblasts in a first embodiment of the present invention;

FIG. 4 is a graph showing the results of evodiamine inhibiting extracellular matrix secretion of scar fibroblasts in a first embodiment of the present invention;

FIG. 5 is a graph showing the results of inhibition of cytoskeleton of scar fibroblasts by cornearine in example one of the present invention;

FIG. 6 is a graph showing the results of animal experiments in the second embodiment of the present invention in which evodiamine was evaluated for inhibition of scar.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the following examples are specifically set forth in the following embodiments in combination with the accompanying drawings.

< example one >

This example is an in vitro experimental study of evodiamine to inhibit the biological activity of scar muscle fibroblasts.

1.1 Experimental methods

(1) Scar fibroblast isolation

Patients agree and sign an informed consent, hypertrophic scar tissue is obtained in the operation, after repeated PBS cleaning, the hypertrophic scar tissue is cut into tissue blocks with the size of about 1cm, dispase is digested at 4 ℃ overnight, the epidermis is stripped, the dermis part is digested for about 1 hour by using 0.2% collagenase, suspension is filtered by using a 200-mesh filter screen, then the suspension is collected and centrifuged, the complete culture medium is subjected to suspension culture, and the next experiment is carried out by using the 2 nd-3 rd generation cells.

(2) CCK-8 method for detecting proliferation of scar fibroblast

Cells were plated in 96-well plates (about 8000 cells/well) and cultured in complete medium for 12 hours before addition of various concentrations of drug. The experiment was divided into four groups: 0 μ M evodiamine group, 0.05 μ M evodiamine group, 0.25 μ M evodiamine group, and 1.25 μ M evodiamine group. When cell proliferation is detected, 10 mu l of CCK-8 reagent is added into each hole, and after incubation and culture for 3 hours in a dark place, the OD value of each hole under the absorbance of 450nm is detected by using a microplate reader.

(3) Detection of apoptosis and cycle by annexin V/PI flow cytometry

Cells were plated in 96-well plates (approximately 5X 10)⁵Individual cells/well), various concentrations of drug were added after 12 hours of culture in complete medium. The experiment was divided into four groups: 0 μ M evodiamine group, 0.05 μ M evodiamine group0.25 μ M evodiamine group, 1.25 μ M evodiamine group. After further incubation for 24 hours the cells were digested into a single cell suspension and resuspended in 100. mu.L of buffer. When detecting apoptosis, 5 mu of LFITC-annexin V and 10 mu of L of Propidium Iodide (PI) are added to incubate for 10 minutes at room temperature, and a Beckman flow cytometer is adopted to detect the proportion of apoptotic cells; when detecting cell proliferation, 500. mu.L of DNA staining solution and 5. mu.L of cell penetrating solution are added, and after incubation for 30 minutes, the machine is operated for detection.

(4) Extracellular matrix expression assay

Cells were plated in 96-well plates (approximately 5X 10)⁵Individual cells/well), and adding drugs with different concentrations after the cells are fused to 70% -80%. The experiment was divided into four groups: 0 μ M evodiamine group, 0.05 μ M evodiamine group, 0.25 μ M evodiamine group, and 1.25 μ M evodiamine group. Expression of type I collagen (collagen I), type III collagen (collagen III) and smooth muscle actin (α -SMA) was detected after 24 hours using a western-blot. The method comprises the following specific steps: 100-150 μ L of protein lysate was added to each well of the 6-well plate, centrifuged at 12000g for 15 min at 4 ℃ after 20 min, and then the protein was boiled for 5 min before loading. Sealing the membrane by 5 percent of skimmed milk powder for one hour at room temperature after electrophoresis and membrane conversion, respectively adding corresponding primary antibodies, incubating overnight at 4 ℃, adding corresponding secondary antibodies, and finally developing by using a Horse Radish Peroxidase (HRP) -ECL luminescence method.

1.2 statistical analysis

The statistical analysis adopts software SPSS 20.0, the comparison between multiple groups of data of counting data adopts ANOVA analysis, the comparison between two groups of data adopts t test, and the difference is considered to have statistical significance when P is less than 0.05.

1.3 results of the experiment

FIG. 1 is a general view of evodiamine induced apoptosis of scar fibroblasts in a first embodiment of the present invention; FIG. 2 is a flow chart of the results of evodiamine induced apoptosis of scar fibroblasts in accordance with the first embodiment of the present invention; FIG. 3 is a graph showing the results of evodiamine inhibiting proliferation of scar fibroblasts in a first embodiment of the present invention; FIG. 4 is a graph showing the results of evodiamine inhibiting extracellular matrix secretion of scar fibroblasts in a first embodiment of the present invention; FIG. 5 is a graph showing the results of inhibition of cytoskeleton of scar fibroblasts by cornearine in example one of the present invention.

As shown in figures 1-5, after 24 hours of action of evodiamine with different concentrations, massive apoptosis of scar fibroblasts can be seen, cells are round, bright and even floating, and the effect is gradually enhanced along with the increase of the drug concentration, and the apoptosis is increased when the concentration is higher (figure 1). The Annexin V/PI flow cytometry detection further proves the general apoptosis result, and the result shows that the effect of the evodiamine on the scar fibroblasts is concentration-dependent, and the apoptosis is more obvious when the concentration is higher (figure 2). The CCK-8 test detects cell proliferation, and finds that the minimum concentration (0.05 mu M) of evodiamine has obvious inhibition effect on cell proliferation of scar fibroblasts, and the difference is statistically different from a control group after the medicine acts for 24 hours and is more obvious when the medicine acts for a longer time (figure 3A). Further, the cell cycle was examined by flow cytometry, and it was found that evodiamine inhibited cells at the G2/M phase, and the inhibition effect was more significant with increasing concentration (FIGS. 3B-C). We adopt western blot to detect the influence of evodiamine with different concentrations on the secretion of extracellular matrix of scar fibroblasts, and the results show that the evodiamine can inhibit the expression of type I collagen (collagen I), type III collagen (collagen III) and smooth muscle actin (alpha-SMA), the inhibition is concentration-dependent, and the inhibition is more obvious when the medicine concentration is higher (figure 4). In order to explore the action mechanism of evodiamine for inhibiting the biological activity of scar fibroblast, the arrangement of microtubule microfilaments of cells is further detected. The alpha-tubulin (alpha-tubulin) immunofluorescence result shows that the microtubule network of the scar fibroblast in the control group is arranged normally and extends uniformly from the cell nucleus to the cell periphery; after 24 hours of evodiamine, microtubules in the cytoplasm accumulated around the nucleus, and the morphology of the cells changed from a long spindle to a blunt and irregular shape (FIG. 5A). F-actin is specifically stained by immunofluorescence of Phalloidin (Phalloidin), and the microfilament is obviously disturbed and shortened after the evodiamine treatment, and the cell morphology is also obviously changed (figure 5B).

< example two >

This example is an animal experiment to evaluate the inhibitory effect of evodiamine on scarring.

2.1 Rabbit ear scar model establishment

8 male New Zealand rabbits are purchased from the experimental animal center of naval military medical university, and have the size of 2-3 months and the weight of 2.0-2.5 kg. After success of ear margin intravenous injection anesthesia, 75% alcohol is adopted for disinfection, 6 round full-layer skin defect wound surfaces with the diameter of 1cm are prepared on the ventral side of the ear of each rabbit, and the full-layer skin, subcutaneous tissues and perichondrium are carefully separated and removed. The drug for sedation and analgesia is given after the operation, and the wound surface is observed every day until the wound surface is completely epithelialized 3 weeks after the operation.

2.2 Experimental groups

The 8 new zealand rabbits were randomly divided into 2 groups: the experimental group is that the evodiamine is injected into scar locally, the concentration of the medicine is 50 mu M, each wound surface is 100 mu L, and the injection is carried out once in 3 days and 6 times in total; the control group was injected with 100 μ LPBS locally per wound surface, once every 3 days, for a total of 6 injections.

2.3 evaluation of the experiment

(1) General aspect of scar

Taking pictures every week after operation to observe the hardness, color and softness of the scar.

(3) Histological examination

The materials are taken on the 39 th day after operation, each scar tissue is divided into 2 parts, one part is used for western blot detection of expression of collagen I, collagen III and alpha-SMA (the experimental method is the same as example 1), the other part is fixed by 4% paraformaldehyde and then embedded by paraffin, and the tissue section is subjected to H & E staining and MASSON staining conventionally to observe the collagen formation condition in the scar tissue.

2.4 statistical analysis

The statistical analysis adopts software SPSS 20.0, the comparison between multiple groups of data of counting data adopts ANOVA analysis, the comparison between two groups of data adopts t test, and the difference is considered to have statistical significance when P is less than 0.05.

2.5 results of the experiment

FIG. 6 is a graph showing the results of animal experiments in the second embodiment of the present invention in which evodiamine was evaluated for inhibition of scar.

As shown in fig. 6, the wound surface was completely epithelialized 3 weeks after the operation, and the control group was seen to have thick and hard scar, deep red color and convex skin surface on day 39 after the operation; the scar of the evodiamine administration group is obviously improved compared with the control group in aspects of hardness, thickness, color and the like (figure 6A). H & E staining was used to calculate the scar thickness values, and it was found that the scar thickness was significantly reduced in the evodiamine-administered group (FIG. 6B). Further, MASSON staining revealed that the collagen fibers of the evodiamine-administered group were aligned and sparse, while the collagen fibers of the control group were densely and disorganized (fig. 6C). Finally, western blot was used to detect the expression of collagen I, collagen III and α -SMA in scar tissue, and it was found that each index in the evodiamine administration group was significantly reduced, and the difference was statistically significant compared to the control group (fig. 6D).

Effects and effects of the embodiments

The experimental results of the first example and the second example prove that the evodiamine can prevent and/or treat scars by inhibiting the secretion of extracellular matrix of scar muscle fibroblasts, inhibiting the proliferation of scar fibroblasts, promoting the apoptosis of the scar fibroblasts and inhibiting the activity of cytoskeleton of the scar fibroblasts.

The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.