Application of hypericin in the preparation of drugs for pathological scar

Abstract

The present invention provides an application of hypericin in the preparation of a drug for pathological scars, which relates to the field of biotechnology. The application includes applying hypericin to the preparation of a drug for treating and / or preventing pathological scars in a subject; and / or, preparing a drug for down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject; and / or, down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject for non-diagnostic and therapeutic purposes. The application provides an effective ingredient of a safe, reliable, and easy-to-prepare drug for treating and / or preventing skin pathological scars with definite curative effects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the use of hypericin in the preparation of a medicament for pathological scars. Background Art

[0002] The following statements only provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Skin scar is an inevitable product of the body's inability to fully achieve tissue regeneration after skin injury, and then initiate the tissue repair process, which is replaced by connective tissue. Therefore, skin scar is a fibrotic hyperplastic disease with abnormal morphological appearance and functional changes. However, some skin injuries fail to achieve the outcome of "normal" scars, but form pathological scars such as hypertrophic scars and keloids. Pathological scar is an abnormal wound healing outcome; so far, the mechanism that determines the outcome of skin wound healing as a fine "normal" scar, a prominent hypertrophic scar or a tumor-like keloid is still unclear. Pathological scars have the important characteristic that the scar is significantly higher than the skin surface. In addition, hypertrophic scars do not exceed the original injury range; while horizontal growth beyond the original wound edge is a typical feature of keloids. Pathological features such as more fibroblasts, obvious angiogenesis and a large amount of collagen deposition can be seen in the dermis of pathological scars. The main signaling pathways related to the pathogenesis of pathological scars include signaling pathways such as TGFβ.

[0004] With the increase in the number of skin trauma patients and people's demand for cure and beauty, new challenges have been posed for the treatment of pathological scars. There are already some treatment methods for pathological scars, including pressure therapy, drug therapy, etc. Pressure therapy can slightly flatten the scar and relieve itching and pain. However, this method requires long-term adherence, usually for several months or even years, which is difficult for patients to accept. Silicone gel or sheets can soften scar tissue and inhibit scar proliferation. However, the effect varies from person to person, and long-term use is also required. Drug therapy, such as oral or injection of steroid drugs, has a certain effect, which can reduce inflammation and inhibit collagen synthesis. However, there are obvious side effects, such as thinning and atrophy of the skin. Radiation therapy can inhibit scar proliferation, but there are certain side effects, such as skin damage, and it is usually only considered when other treatments are ineffective. To sum up, the existing treatment products for pathological scars in the prior art all have some side effects and poor treatment effects. Therefore, there is an urgent need to develop new treatment products for pathological scars with small toxic and side effects and high effectiveness.

[0005] In view of this, the present invention is particularly proposed. Summary of the Invention

[0006] The object of the present invention is to provide the application of hypericin in the preparation of a drug for pathological scars, so as to alleviate the defects of poor treatment effect and / or side effects of existing pathological scar treatment products in the prior art.

[0007] In the first aspect, there is provided the application of hypericin in one or more of (I) to (IV):

[0008] (I) preparing a drug for treating and / or preventing pathological scars in a subject;

[0009] (II) preparing a drug for treating and / or preventing non-pathological scars in a subject;

[0010] (III) preparing a drug for down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject;

[0011] (IV) down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject for non-diagnostic and therapeutic purposes.

[0012] In the second aspect, there is provided a method for down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject for non-diagnostic and therapeutic purposes, the method comprising administering hypericin to the subject.

[0013] In the third aspect, there is provided a pharmaceutical composition, which comprises hypericin and a pharmaceutically acceptable excipient; the concentration of hypericin in the pharmaceutical composition is at least 2.5 μM, at least 5 μM, at least 10 μM or at least 20 μM.

[0014] In the fourth aspect, there is provided a preparation method of the pharmaceutical composition described in the third aspect, the preparation method comprising mixing hypericin with a pharmaceutically acceptable excipient.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Through molecular docking and computer virtual screening techniques, the present invention discovers that hypericin can effectively bind to key target proteins in the TGFβ signaling pathway, which is a key factor in the formation of pathological scars. Hypericin has significant specificity for the TGFβ signaling pathway and has little effect on other signaling pathways, so its potential side effects are small. As a natural product, hypericin is not only inexpensive but also easy to extract. Experimental results show that hypericin can reduce the expression of collagen in fibroblasts and inhibit the proliferation of fibroblasts in pathological scars by inhibiting the TGFβ signaling pathway. In addition, animal experiments further confirm the effectiveness of hypericin in alleviating pathological scars. Description of the Drawings

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Immunoblotting detection results of type I collagen (collagen I), type III collagen (collagen III), and p-SMAD2 after treating skin fibroblasts in each experimental group in Example 2;

[0019] Figure 2 Cell photos of Transwell results after treating normal skin fibroblasts stimulated by TGFβ with different concentrations of hypericin in Example 3;

[0020] Figure 3 Statistical results of Transwell results after treating normal skin fibroblasts stimulated by TGFβ with different concentrations of hypericin in Example 3;

[0021] Figure 4 Photos of the results of the cell migration experiment in Example 3;

[0022] Figure 5 Dose-effect curve of hypericin on keloid fibroblasts KF1 in Example 4;

[0023] Figure 6 Dose-effect curve of hypericin on keloid fibroblasts KF2 in Example 4;

[0024] Figure 7 Photos of the wound over time after applying drugs to the wounds of the animal models in each experimental group in Example 5;

[0025] Figure 8 Relative scar sizes of the animal models in each experimental group on the 30th day after applying drugs to the wounds of the animal models in each experimental group in Example 5. Specific Embodiments

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0027] As used herein, "and / or" is used to indicate that one or both of the stated situations may occur; for example, A and / or B includes (A and B) and (A or B).

[0028] As used herein, unless otherwise specified, any numbering is used to distinguish one entity or act from another, rather than necessarily requiring or implying any actual such relationship, order, or degree of importance between these entities or acts; for example, numbers I......III; first, second... fourth, etc.

[0029] As used herein, unless otherwise specified, "optionally", "optional", "alternative", or "alternatively" means that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur.

[0030] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps.

[0031] As used herein, the term "pathological scar" refers to a benign fibrotic hyperplastic disease that appears after the dermis is damaged due to trauma, infection, etc. Clinically, it often presents as a red nodular elevation that affects the appearance and may be accompanied by discomfort such as itching and stinging. It usually includes two types: hypertrophic scar and keloid. "Non-pathological scar" refers to other types of scars excluding "pathological scar", such as physiological scar.

[0032] As used herein, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition is to be administered.

[0033] As used herein, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, alpacas, birds, goats, and sheep. In certain embodiments, the subject is a human.

[0034] As used herein, the terms "treat", "alleviate", or "ameliorate" are used interchangeably. These terms refer to methods of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or improving one or more diseases, disorders, or conditions associated with the underlying disorder being treated.

[0035] As used herein, the terms "prevent" and "preclude" are used interchangeably, i.e., they refer to methods of obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit", a drug may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease has not yet been made.

[0036] As used herein, the term "effective amount" refers to a therapeutically effective amount, an amount sufficient to reduce or ameliorate the severity and / or duration of a disorder and / or one or more of its symptoms; prevent the progression of a disease; cause the regression of a disease; prevent the recurrence, development or progression of one or more symptoms associated with a disease; or enhance or improve the prophylactic or therapeutic effect of another therapy. Those skilled in the art can proportionally reduce or increase the dosage according to the physiological conditions or requirements of the subject.

[0037] As used herein, a functional derivative refers to a compound obtained by structurally modifying or functionally transforming a parent compound (such as a natural product, a bioactive molecule or a synthetic compound) by chemical, physical or biological methods. For example, by introducing new functional groups, changing the molecular conformation or binding to other molecules, on the basis of maintaining the core structure and function of the parent compound, a compound with enhanced or expanded partial functions is obtained. Exemplary functional derivatives include, for example, compounds obtained by introducing new functional groups into the parent compound, such as esterification, acylation or sulfonation; or compounds formed by reacting with acids or bases to form salts, etc.

[0038] Hypericin is derived from the natural medicine Hypericum perforatum L., and is the most biologically active component therein. The original plant is mainly distributed in provinces such as Zhejiang and Fujian in China, as well as in the southwest and northwest regions. It is rich in resources and has a large output in China. The molecular formula of hypericin: C 30 H 16 O8, and its chemical name is 4,4',5,5',7,7'-hexahydroxy-2,2'-dimethyl-meso-naphthacenedione, and its structure is shown in the molecular formula (i). As used herein, unless otherwise specified, hypericin also includes its functional derivatives.

[0039] Molecular formula (i).

[0040] Hypericin has a delicate fragrance and is soluble in alkaline aqueous solutions, pyridine, organic amines, etc. Studies have shown that hypericin has antiviral, antidepressant and anti-tumor effects, etc. The present invention discovers that hypericin can down-regulate the collagen expression of fibroblasts by inhibiting TGFβ, and can inhibit the proliferation of primary cultured fibroblasts of pathological scars. Verified by animal experiments, hypericin can relieve pathological scars. Based on this discovery, the following technical solutions are provided.

[0041] In a first aspect, there is provided the use of hypericin in one or more of (I) to (IV):

[0042] (I) Preparing a drug for treating and / or preventing pathological scars in a subject;

[0043] (II) Preparing a drug for treating and / or preventing non-pathological scars in a subject;

[0044] (III) Preparing a drug for downregulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein in a subject;

[0045] (IV) Downregulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein (p-SMAD2) in a subject for non-diagnostic and non-therapeutic purposes.

[0046] In an alternative embodiment, the pathological scar includes hypertrophic scar and keloid.

[0047] In an alternative embodiment, hypericin is used in the preparation of a drug for downregulating type I collagen, type III collagen, and phosphorylated SMAD2 protein in a subject.

[0048] In an alternative embodiment, hypericin is used for downregulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein in a subject for non-diagnostic and non-therapeutic purposes.

[0049] In an alternative embodiment, the working concentration of hypericin in the application is at least 2.5 μM, at least 5 μM, at least 10 μM, or at least 20 μM. The working concentration is the working concentration of hypericin in the drug described in any one of (I)-(III); or the concentration of hypericin administered to the subject when downregulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein in (IV).

[0050] In an alternative embodiment, hypericin in the application is delivered by a delivery formulation, and the delivery formulation includes, but is not limited to, one or more of lipids (such as ionizable lipids, phospholipids, structural lipids (such as cholesterol), PEG lipids), nanoparticles, proteins (such as cationic proteins or antibodies or antigen-binding fragments thereof), polypeptides (such as cationic polypeptides), cationic polymers, nucleic acid aptamers, or small molecule compounds.

[0051] In an alternative embodiment, hypericin in the application is delivered by liposomes, lipid nanoparticles, lipid vesicles, or lipid complexes, preferably delivered by liposomes. Liposomes are artificially synthesized vesicular microparticles composed of a phospholipid bilayer, and their structure is similar to the cell membrane. They can encapsulate various substances, such as drugs, proteins, DNA, RNA, etc., and transport them to specific parts of the body. They are a widely used carrier in the biomedical field, stable, safe, easy to use, and have a mature production process. Using liposomes can replace potentially toxic solvents such as DMSO when using hypericin, avoid the toxic side effects caused by toxic solvents, and significantly enhance the safety and therapeutic effect of the drug.

[0052] In an alternative embodiment, hypericin in the application is delivered by liposomes composed of distearoyl phosphatidylethanolamine - polyethylene glycol.

[0053] In an alternative embodiment, the drug is a topical preparation.

[0054] In an alternative embodiment, the concentration of hypericin in the topical preparation is 1 - 1000 μg / ml, such as but not limited to 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 μg / ml, preferably 50 μg / ml.

[0055] In an alternative embodiment, the subject includes mammals.

[0056] In an alternative embodiment, the subject includes fibroblasts; further optionally, the fibroblasts are fibroblasts derived from mammals; and / or, further optionally, the fibroblasts are fibroblasts derived from keloid, a pathological scar.

[0057] In an alternative embodiment, hypericin is used as the sole active ingredient in the application of any one of (I) - (IV).

[0058] In an alternative embodiment, hypericin is used in combination with other active ingredients in the application of any one of (I) - (IV). When hypericin is used in combination with one or more other active ingredients, it can be administered simultaneously with the one or more other active ingredients. In some such embodiments, hypericin and the other active ingredients can be administered simultaneously as part of the same composition. In other embodiments, hypericin "used in combination" with other active ingredients does not need to be administered simultaneously or in the same composition as the active ingredient. That is, one or more active ingredients administered after and / or before hypericin in the present invention are also considered to be "used in combination" with hypericin, even if hypericin and other active ingredients are administered by different routes.

[0059] In a second aspect, a method for down - regulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein in a subject for non - diagnostic and non - therapeutic purposes is provided, the method comprising administering hypericin to the subject.

[0060] In an alternative embodiment, administering hypericin to the subject includes contacting hypericin with the subject.

[0061] In an alternative embodiment, administering hypericin to the subject includes using a delivery preparation to deliver hypericin into the subject's cells.

[0062] In an alternative embodiment, the subject includes fibroblasts; further optionally, the fibroblasts are fibroblasts derived from a mammal; and / or, further optionally, the fibroblasts are derived from fibroblasts of a pathological scar, keloid.

[0063] In an alternative embodiment, administering hypericin to the subject includes culturing fibroblasts in a culture medium containing an effective amount of hypericin.

[0064] In a third aspect, a pharmaceutical composition is provided, which is used for treating and / or preventing pathological scars of a subject; or, for downregulating at least one of type I collagen, type III collagen, and phosphorylated SMAD2 protein in a subject. The pharmaceutical composition contains hypericin and a pharmaceutically acceptable excipient; the concentration of hypericin in the pharmaceutical composition is at least 2.5 μM, at least 5 μM, at least 10 μM, or at least 20 μM.

[0065] In an alternative embodiment, the pharmaceutical composition contains a delivery preparation for delivering hypericin, and the delivery preparation includes, but is not limited to, one or more of lipids (such as ionizable lipids, phospholipids, structural lipids (such as cholesterol), PEG lipids), nanoparticles, proteins (such as cationic proteins or antibodies or antigen-binding fragments thereof), polypeptides (such as cationic polypeptides), cationic polymers, nucleic acid aptamers, or small molecule compounds.

[0066] In an alternative embodiment, hypericin in the pharmaceutical composition is loaded in at least one of liposomes, lipid nanoparticles, lipid vesicles, and lipid complexes.

[0067] In an alternative embodiment, hypericin in the pharmaceutical composition is loaded in liposomes formed by stearoyl phosphatidylethanolamine - polyethylene glycol.

[0068] In an alternative embodiment, the stearoyl phosphatidylethanolamine - polyethylene glycol includes stearoyl phosphatidylethanolamine - polyethylene glycol 2000.

[0069] In an alternative embodiment, the feeding ratio of hypericin to stearoyl phosphatidylethanolamine - polyethylene glycol 2000 is 1:(1 - 10), for example, but not limited to, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, and preferably 1:5.

[0070] In alternative embodiments, the pharmaceutically acceptable excipients include, but are not limited to, one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, bacteriostatic agents, suspending agents, coating agents, film-forming agents, fragrances, thickening agents, anti-adhesives, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickening agents, clathrates, protectants, humectants, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrices, and drug carrier materials. Those skilled in the art can select the composition and dosage of the excipients according to the required dosage form and general technical information in the art, such as textbooks, reference documents, process manuals, product descriptions, standard documents, etc., and the present invention does not limit this.

[0071] In alternative embodiments, the pharmaceutical composition is a topical preparation. The topical preparation includes, but is not limited to, ointments, gels, paints, patches, or films.

[0072] In alternative embodiments, the pharmaceutical composition is a topical preparation, and the topical preparation contains liposomes, lipid nanoparticles, lipid vesicles, or lipid complexes loaded with hypericin; and a matrix, and the matrix can be, but is not limited to, a solvent, a gel, an ointment, or a film.

[0073] In alternative embodiments, the pharmaceutical composition is a topical preparation, and the topical preparation contains liposomes loaded with hypericin, and the liposomes are composed of distearoyl phosphatidylethanolamine-polyethylene glycol. Optionally, the matrix is a solvent.

[0074] In alternative embodiments, the concentration of hypericin in the topical preparation is 1 to 1000 μg / ml, such as, but not limited to, 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg / ml, and preferably 50 μg / ml.

[0075] In a fourth aspect, a method for preparing the pharmaceutical composition described in the third aspect is provided, and the preparation method includes mixing hypericin with pharmaceutically acceptable excipients.

[0076] In alternative embodiments, the pharmaceutical composition includes dropping a first solution into a second solution, stirring the second solution during the dropping process; continuing to stir the mixture after the dropping is completed; and then removing the solvents of the first solution and the second solution from the mixture; the first solution contains hypericin; and the second solution contains lipid components for forming liposomes.

[0077] In an alternative embodiment, the solvent of the first solution is an organic phase, such as, but not limited to, ethanol, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol or ethyl acetate, preferably DMSO.

[0078] In an alternative embodiment, the solvent of the second solution is an aqueous phase, and the aqueous phase may be a buffered or unbuffered solution; in an alternative embodiment, the solvent of the second solution is PBS.

[0079] In an alternative embodiment, after the dropping is completed, the mixture is continuously stirred for 3 to 5 h, preferably 4 h.

[0080] In an alternative embodiment, the solvents of the first solution and the second solution in the mixture are removed by dialysis.

[0081] In an alternative embodiment, the lipid component includes stearoyl phosphatidylethanolamine-polyethylene glycol, preferably includes stearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0082] In an alternative embodiment, the feeding ratio of hypericin to stearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 1:(1-10), such as, but not limited to, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, preferably 1:5.

[0083] In a specific embodiment, the preparation method of the pharmaceutical composition is as follows:

[0084] (a) Weigh 5 mg of hypericin and dissolve it in 500 μl of DMSO, and mix evenly to obtain solution A.

[0085] (b) Weigh 25 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and dissolve it in 2.5 ml of PBS, and mix evenly to obtain solution B.

[0086] (c) Slowly and evenly drop solution A into solution B, continuously stir while adding, and then continue to stir and react for 4 hours.

[0087] (d) Collect the above mixed solution into a dialysis bag and dialyze it with PBS solution for 48 hours to remove DMSO and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 that has not formed liposomes.

[0088] (e) The above dialysis membrane was made up to 5 ml with PBS to obtain the required stock solution of hypericin-liposome solution with a concentration of 1 mg / ml.

[0089] In an alternative embodiment, the hypericin in any one of the first, second, third, and fourth aspects is hypericin as shown in formula (i) with a CAS number of 548-04-9.

[0090] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0091] The hypericin in Examples 1 to 5 is hypericin as shown in formula (i) with a CAS number of 548-04-9.

[0092] Example 1

[0093] For the molecular docking and computer-aided virtual screening of the key target proteins in the TGFβ signaling pathway, which is the key pathway for the pathogenesis of pathological scars, small molecule natural products (Table 1) screened from the library were used. Molecular docking showed that hypericin had a low energy when binding to TGFBR1, indicating that hypericin could effectively bind to TGFBR1.

[0094] Table 1 Molecular docking results with TGFBR1 as the target

[0095]

[0096] Example 2

[0097] 10 mg of accurately weighed hypericin powder was dissolved in 1.9824 ml of DMSO and stirred thoroughly until the solution was clear to obtain a 10 mM hypericin-DMSO stock solution.

[0098] The above hypericin-DMSO stock solution was added to the medium of normal skin fibroblasts stimulated with TGFβ according to the dosage, so that the final concentrations of hypericin in the medium were 0, 2.5, 5, 10, and 20 μM respectively. After 48 hours, the proteins of the cells were collected, and the expression levels of type I collagen and type III collagen related to the pathogenesis of pathological scars, and the expression level of p-SMAD2 related to the activation of the TGFβ pathway were detected by Western Blot experiment. It was found that the expressions of type I collagen, type III collagen, and p-SMAD2 in fibroblasts were significantly enhanced after TGFβ stimulation, while their expressions were significantly reduced after hypericin treatment, and the degree of reduction was positively correlated with the hypericin concentration ( Figure 1), The above experimental results indicate that hypericin can inhibit the expression of type I collagen, type III collagen, and p-SMAD2.

[0099] Conclusion: In the above cell experiments, the results show that hypericin can downregulate collagen expression in fibroblasts by inhibiting TGFβ.

[0100] Example 3

[0101] Resuspend normal fibroblasts in serum-free medium and add them to the upper chamber of a transwell insert. The total number of cells is 500 cells / well, and the volume is 600 μl. At the same time, add the above hypericin-DMSO mother liquor to the upper chamber medium according to the dose, so that the final concentration of hypericin in the medium is 0, 2, and 4 μM respectively, and add TGFβ stimulation. Add 800 μl of normal medium to the lower chamber of the transwell insert. After 24 hours, remove the insert, wipe off the cells in the upper chamber, and stain the cells in the lower chamber with crystal violet, take pictures and count the number of cells in each field of view. This number of cells is the number of cells that have passed through the transwell membrane in the field of view. The results are as Figure 2 and Figure 3 shown. It was found that the migration ability of fibroblasts was enhanced after TGFβ stimulation, while their migration ability was significantly reduced after treatment with hypericin.

[0102] Cultivate normal skin fibroblasts to a confluence of 80-90%, use a sterile pipette tip or a scratching tool to make a scratch on the cell monolayer, and remove the floating cells; culture them with media containing hypericin concentrations of 0, 2, and 4 μM respectively, and add TGFβ stimulation. Take pictures at 0, 24, and 48 h. The results are as Figure 4 shown. It can be seen that hypericin can effectively inhibit the migration of TGFβ-stimulated skin fibroblasts.

[0103] Conclusion: In the above cell experiments, the results show that hypericin can inhibit the migration of fibroblasts induced by TGFβ.

[0104] Example 4

[0105] Add the hypericin-DMSO mother liquor in Example 2 to the media of primary cultured fibroblasts from 2 cases of keloid (named KF1 and KF2 respectively) of pathological scars according to the dose, so that the final concentration of hypericin in the KF1 medium is 0, 2.5, 5, 10, and 20 μM respectively, and the final concentration in the KF2 medium is 0, 2, 4, 8, and 16 μM respectively. After 48 hours, detect the cell viability by CCK8 assay (compared with the group with a hypericin concentration of 0), and make a dose-effect curve. The results show that as the concentration of hypericin increases, the viability of keloid fibroblasts decreases significantly ( Figure 5 and Figure 6), The half-inhibitory concentrations (IC50) of hypericin against KF1 and KF2 were 10.33 μM and 10.28 μM, respectively, indicating that hypericin can inhibit the proliferation of pathological scar fibroblasts.

[0106] Conclusion: The above cell experiments showed that hypericin can inhibit the proliferation of primary cultured fibroblasts in pathological scars.

[0107] Example 5

[0108] Preparation of hypericin-DMSO stock solution: Weigh accurately 10 mg of hypericin powder and dissolve it in 10 ml of DMSO. Stir well until the solution is clear to obtain a 1 mg / ml hypericin-DMSO stock solution.

[0109] Preparation of hypericin-liposome solution stock solution: Weigh accurately 10 mg of hypericin powder and dissolve it in 1 ml of DMSO to obtain a homogeneous solution A. Weigh 50 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 and dissolve it in 5 ml of PBS to obtain a homogeneous solution B. Slowly and evenly add solution A to solution B while continuously stirring, and then continue stirring and reacting for 4 hours. Collect the above mixed solution into a dialysis bag and dialyze it with PBS solution for 48 hours to remove DMSO and polyethylene glycol that did not form liposomes. Filter the above dialysis solution through a membrane and make up the volume to 10 ml with PBS to obtain the required hypericin-liposome solution stock solution with a concentration of 1 mg / ml (calculated based on the content of hypericin).

[0110] The hypericin solutions prepared by the two methods were used for animal experimental studies, and the experimental protocol was as follows: The above-mentioned hypericin-DMSO mother liquor and hypericin-liposome solution mother liquor were respectively diluted with PBS to 50 μg / ml. Proliferative scar models were established on the ears of rabbits. The modeling method was as follows: The rabbits were fixed in a fixator, the head was exposed, and the hair was removed with a hair remover. The skin was disinfected with an alcohol cotton ball, and the rabbits were anesthetized by slow intravenous injection of 5 mg / kg of propofol or continuous inhalation of isoflurane. After successful anesthesia, circular wounds were drilled at the selected sites with a skin trephine, with each wound spaced 1.5 - 2 cm apart and reaching the cartilage surface. The skin was removed with forceps and scissors, and the perichondrium was removed with a scalpel and scissors, while the cartilage was retained. Erythromycin ointment was given after the operation to prevent wound infection. Bleeding and secretion were observed within 3 days after the operation, and then the rabbits were allowed to drink water and eat freely, and the wounds were allowed to heal naturally. The formation of scar tissue that was significantly higher than the normal skin and did not exceed the wound area was regarded as successful modeling. On the third day after starting the modeling, the rabbits were divided into 3 groups, and 100 μl of 50 μg / ml hypericin-DMSO solution, 50 μg / ml hypericin-liposome solution, or an equal volume of PBS solution was applied to the wounds every day. The scar formation was recorded at different days after drug administration, and the size and color of the scars were analyzed on the 30th day after modeling.

[0111] The experimental results are compared as follows:

[0112] On the 30th day after drug administration, the hypericin + liposome group in the 3 groups had the best effect, with the smallest formed proliferative scar and the lightest scar color, which was closest to the color of the normal skin beside it. Its therapeutic effect was much better than that of the hypericin + DMSO group. The PBS group had the worst effect, with a large area of proliferative scar and a red scar color ( Figure 7 and Figure 8 ).

[0113] Conclusion: From the above comparative experiments, the results show that the hypericin + liposome group has a significantly better therapeutic effect on proliferative scars than the hypericin + DMSO group. Liposomes increase the amount of hypericin penetrating the skin and entering cells, and avoid the toxic and side effects caused by DMSO.

[0114] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of hypericin as the only active ingredient in the preparation of a drug for treating and / or preventing pathological scars in a subject.

2. The application according to claim 1, wherein The working concentration of hypericin is at least 2.5 μM, at least 5 μM, at least 10 μM or at least 20 μM.

3. The application according to claim 1 or 2, characterized in that, In the above application, hypericin is delivered by a delivery preparation.

4. The application according to claim 3, wherein The delivery preparation includes liposomes, nanoparticles, proteins, polypeptides, cationic polymers, nucleic acid aptamers or small molecule compounds.

5. The application according to claim 1, wherein, The drug is a topical preparation.

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