Application of hypericin in preparation of medicine for pathological scars

Abstract

The invention provides application of hypericin in preparation of a medicine for pathological scars, and relates to the technical field of biology. According to the application, hypericin is applied to preparation of the medicine for treating and / or preventing pathological scars of subjects. And / or preparing a drug for down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein of a subject; and / or down-regulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in the subject for non-diagnostic and therapeutic purposes. The application provides an effective component which is safe and reliable, has a definite curative effect, is easy to prepare and is used for treating and / or preventing dermatologic scars.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of hypericin in preparing medicine for pathological scars. Background Art

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

[0003] Skin scars are the inevitable product of the body's inability to completely regenerate tissue after skin damage, and the initiation of the tissue repair process, which is replaced by connective tissue. Therefore, skin scars are fibroproliferative diseases with abnormal morphology and functional changes. However, some skin injuries fail to achieve the outcome of "normal" scars, but instead form pathological scars such as hypertrophic scars and keloids. Pathological scars are abnormal wound healing outcomes; to date, the mechanism that determines whether the outcome of skin wound healing is a small "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 will not extend beyond the original injury range; while horizontal growth beyond the edge of the original wound is a typical feature of keloids. Pathological scars have pathological characteristics such as more fibroblasts, obvious angiogenesis, and a large amount of collagen deposition in the dermis. The main signaling pathways associated with the onset 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 healing and beauty, new challenges have been raised for the treatment of pathological scars. There are some treatments for pathological scars, including pressure therapy, drug therapy, etc. Pressure therapy can slightly flatten scars and relieve itching and pain. However, this method requires long-term persistence, usually months or even years, which is difficult for patients to accept. Silicone gel or sheet can soften scar tissue and inhibit scar proliferation. However, the effect varies from person to person, and long-term persistence 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. Radiotherapy can inhibit the proliferation of scars, but there are certain side effects, such as skin damage, and it is usually only considered when other treatments are ineffective. In summary, the treatment products for pathological scars in the prior art all have some side effects and poor treatment effects. Therefore, it is urgent to develop new pathological scar treatment products with small toxic and side effects and effective.

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

[0006] The purpose of the present invention is to provide the use of hypericin in the preparation of a medicament for pathological scars, so as to alleviate the defects of the prior art products for treating pathological scars, such as poor therapeutic effects and / or the presence of side effects.

[0007] In the first aspect, the invention provides the use of hypericin in one or more of (I) to (IV): (I) preparing a drug for treating and / or preventing pathological scars in a subject; (II) preparing a medicament for treating and / or preventing non-pathological scars in a subject; (III) preparing a drug for downregulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject; (IV) Downregulation of at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in subjects for non-diagnostic and non-therapeutic purposes.

[0008] In a second aspect, a method 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 is provided, the method comprising administering hypericin to the subject.

[0009] In a third aspect, a pharmaceutical composition is provided, comprising 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.

[0010] In a fourth aspect, a method for preparing the pharmaceutical composition of the third aspect is provided, the method comprising mixing hypericin with a pharmaceutically acceptable excipient.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses molecular docking and computer virtual screening technology to find 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 relatively 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 confirmed the effectiveness of hypericin in reducing pathological scars. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The immunoblot detection results of type I collagen (collagen I), type III collagen (collagen III) and p-SMAD2 after the skin fibroblasts were treated in each experimental group in Example 2; Figure 2 The cell photos are of the Transwell results of normal skin fibroblasts stimulated by TGFβ after being treated with hypericin at different concentrations in Example 3; Figure 3 The statistical results of Transwell results after treating normal skin fibroblasts stimulated by TGFβ with different concentrations of hypericin in Example 3; Figure 4 This is a photo of the cell migration experiment results in Example 3; Figure 5 is the dose-effect curve of hypericin on keloid fibroblast KF1 in Example 4; Figure 6 is the dose-effect curve of hypericin on keloid fibroblast KF2 in Example 4; Figure 7 These are photos of the changes of the wounds over time after the drugs in each experimental group of Example 5 were applied to the wounds of the animal models; Figure 8 The relative sizes of scars in the animal models of each experimental group on the 30th day after the wounds of the animal models were smeared with the drugs in each experimental group of Example 5. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] As used herein, "and / or" is used to indicate that either or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0016] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between these entities or actions, such as numbering Ⅰ...Ⅲ; first, second...fourth, etc.

[0017] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0018] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0019] In this article, the term "pathological scar" refers to a benign fibroproliferative disease that occurs after the dermis is damaged due to trauma or infection. Clinically, it often manifests as red nodular protrusions that affect the appearance and may be accompanied by discomfort such as itching and tingling. It usually includes two types: hypertrophic scars and keloids. "Non-pathological scars" refer to other types of scars other than "pathological scars", such as physiological scars.

[0020] As used herein, the term "pharmaceutical composition" is in a form that permits the biological activity of the active ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0021] 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.

[0022] As used herein, the terms "treat," "alleviate," or "ameliorate" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of one or more diseases, conditions, or conditions associated with the underlying disorder being treated.

[0023] As used herein, the terms "prevention" and "prevention" are used interchangeably to refer to an approach to obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a drug may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0024] In this article, the term "effective amount" refers to a therapeutic amount that is sufficient to reduce or improve the severity and / or duration of a condition or one or more symptoms thereof; prevent the progression of a disease; cause regression of a disease; prevent the recurrence, development or progression of one or more symptoms associated with a disease; or enhance or improve the preventive or therapeutic effect of another therapy. A person skilled in the art may reduce or increase the dosage in proportion to the subject's physiological condition or requirements.

[0025] In this article, functional derivatives refer to compounds with enhanced or expanded functions obtained by chemical, physical or biological methods to modify the structure or functionalize the parent compound (such as natural products, bioactive molecules or synthetic compounds), for example, by introducing new functional groups, changing the molecular conformation or combining with other molecules, while maintaining the core structure and function of the parent compound. Exemplary functional derivatives include compounds obtained by introducing new functional groups into the parent compound, such as esterification, acylation or sulfonation, or reacting with acids or bases to form salts.

[0026] Hypericin comes from the natural drug Hypericum perforatum and is the most biologically active component. The original plant is mainly distributed in Zhejiang, Fujian and other provinces in my country, as well as in the southwest and northwest. It is rich in resources and has a large output in my country. Hypericin molecular formula: C 30 H 16 O 8 The chemical name is 4,4',5,5'7,7'-hexahydroxy-2,2'-dimethyl-meso-naphtho-dianthrone, and the structure is shown in the molecular formula (i). In this article, unless otherwise specified, hypericin also includes its functional derivatives.

[0027] Molecular formula (I).

[0028] Hypericin has a fresh fragrance and is soluble in alkaline aqueous solution, pyridine, organic amines, etc. Studies have shown that hypericin has antiviral, antidepressant and antitumor effects. The present invention finds that hypericin can downregulate the collagen expression of fibroblasts by inhibiting TGFβ, and can inhibit the proliferation of primary cultured fibroblasts of pathological scars. Animal experiments have verified that hypericin can relieve pathological scars. Based on this discovery, the following technical solution is provided.

[0029] In the first aspect, the invention provides the use of hypericin in one or more of (I) to (IV): (I) preparing a drug for treating and / or preventing pathological scars in a subject; (II) preparing a medicament for treating and / or preventing non-pathological scars in a subject; (III) preparing a drug for downregulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject; (IV) Downregulation of at least one of the following: type I collagen, type III collagen and phosphorylated SMAD2 protein (p-SMAD2) in the subject for non-diagnostic and non-therapeutic purposes.

[0030] In an optional embodiment, the pathological scars include hypertrophic scars and keloids.

[0031] In an optional embodiment, the hypericin is used to prepare a drug for down-regulating type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject.

[0032] In an optional embodiment, the hypericin is used for down-regulating type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject for non-diagnostic and therapeutic purposes.

[0033] In an optional 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) to (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 the subject in (IV).

[0034] In an optional embodiment, hypericin is delivered by a delivery formulation in the application, and the delivery formulation includes but is not limited to one or more of lipids (e.g., ionizable lipids, phospholipids, structural lipids (e.g., cholesterol), PEG lipids), nanoparticles, proteins (e.g., cationic proteins or antibodies or antigen-binding fragments thereof), polypeptides (e.g., cationic polypeptides), cationic polymers, nucleic acid aptamers or small molecule compounds.

[0035] In an optional embodiment, in the application, hypericin is delivered by liposomes, lipid nanoparticles, lipid vesicles or lipid complexes, preferably by liposome delivery. Liposomes are a kind of artificially synthesized vesicle-like microparticles, which are composed of a phospholipid bilayer, and their structure is similar to that of a 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 kind of carrier widely used in the biomedical field, stable and safe, easy to use, and mature production technology. The use of liposomes can replace the potential toxic solvents of hypericin when used, such as DMSO, to avoid the toxic side effects produced by toxic solvents, and significantly enhance the safety and therapeutic effects of drugs.

[0036] In an optional embodiment, in the application, hypericin is delivered by liposomes composed of distearoylphosphatidylethanolamine-polyethylene glycol.

[0037] In an optional embodiment, the drug is an external preparation.

[0038] In an optional embodiment, the concentration of hypericin in the topical preparation is 1 to 1000 μg / ml, for example 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.

[0039] In an alternative embodiment, the subject comprises a mammal.

[0040] In an optional embodiment, the subject comprises fibroblasts; further optionally, the fibroblasts are fibroblasts derived from mammals; and / or, further optionally, the fibroblasts are fibroblasts derived from pathological scars such as keloids.

[0041] In an optional embodiment, hypericin is used as the sole active ingredient in any one of (I) to (IV).

[0042] In an optional embodiment, hypericin is used in combination with other effective ingredients in the application of any one of (I) to (IV). When hypericin is used in combination with one or more other effective ingredients, it can be administered simultaneously with the one or more other effective ingredients. In some such embodiments, hypericin and other effective ingredients can be administered simultaneously as part of the same composition. In other embodiments, hypericin "used in combination" with other effective ingredients does not need to be administered simultaneously or in the same composition as the effective ingredient. That is, in the present invention, one or more effective ingredients administered after and / or before hypericin are also considered to be "used in combination" with hypericin, even if hypericin and the other effective ingredients are administered by different administration methods.

[0043] In a second aspect, a method 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 is provided, the method comprising administering hypericin to the subject.

[0044] In an alternative embodiment, administering hypericin to the subject comprises contacting hypericin with the subject.

[0045] In an alternative embodiment, administering hypericin to a subject comprises delivering hypericin into cells of the subject using a delivery formulation.

[0046] In an optional embodiment, the subject comprises fibroblasts; further optionally, the fibroblasts are fibroblasts derived from mammals; and / or, further optionally, the fibroblasts are fibroblasts derived from pathological scars such as keloids.

[0047] In an alternative embodiment, administering hypericin to the subject comprises culturing fibroblasts in a culture medium containing an effective amount of hypericin.

[0048] In a third aspect, a pharmaceutical composition is provided, the pharmaceutical composition being used for treating and / or preventing pathological scars caused by 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 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.

[0049] In an optional embodiment, the pharmaceutical composition contains a delivery formulation for delivering hypericin, which includes but is not limited to one or more of lipids (e.g., ionizable lipids, phospholipids, structural lipids (e.g., cholesterol), PEG lipids), nanoparticles, proteins (e.g., cationic proteins or antibodies or antigen-binding fragments thereof), polypeptides (e.g., cationic polypeptides), cationic polymers, nucleic acid aptamers or small molecule compounds.

[0050] In an optional embodiment, the hypericin in the pharmaceutical composition is loaded into at least one of liposomes, lipid nanoparticles, lipid vesicles and lipid complexes.

[0051] In an optional embodiment, the hypericin in the pharmaceutical composition is loaded into liposomes formed by stearoylphosphatidylethanolamine-polyethylene glycol.

[0052] In an optional embodiment, the stearoyl phosphatidylethanolamine-polyethylene glycol includes stearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0053] In an optional embodiment, the feed 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, preferably 1:5.

[0054] In an optional embodiment, the pharmaceutically acceptable excipients include, but are not limited to, solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film formers, aromatics, tackifiers, anti-adhesive agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protective agents, humectants, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrix and drug carrier materials. Those skilled in the art can select the composition and dosage of the excipients according to the desired dosage form and general technical information in the art, such as teaching materials, references, process manuals, product descriptions, standard documents, etc., and the present invention does not limit this.

[0055] In an optional embodiment, the pharmaceutical composition is an external preparation, which includes but is not limited to an ointment, a gel, a paint, a patch or a film.

[0056] In an optional embodiment, the pharmaceutical composition is an external preparation, which contains liposomes, lipid nanoparticles, lipid vesicles or lipid complexes loaded with hypericin; and a matrix, which can be but is not limited to a solvent, a gel, a cream or a film.

[0057] In an optional embodiment, the pharmaceutical composition is an external preparation, the external preparation contains liposomes loaded with hypericin, and the liposomes are composed of distearoylphosphatidylethanolamine-polyethylene glycol. Optionally, the matrix is ​​a solvent.

[0058] In an optional embodiment, the concentration of hypericin in the topical preparation is 1 to 1000 μg / ml, for example 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.

[0059] In a fourth aspect, a method for preparing the pharmaceutical composition of the third aspect is provided, the method comprising mixing hypericin with a pharmaceutically acceptable excipient.

[0060] In an optional embodiment, the pharmaceutical composition comprises adding a first solution dropwise to a second solution, stirring the second solution during the adding process; continuing to stir the mixed solution after the adding is completed; then removing the solvent of the first solution and the solvent of the second solution from the mixed solution; the first solution contains hypericin; and the second solution contains lipid components for forming liposomes.

[0061] In an optional 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.

[0062] In an optional embodiment, the solvent of the second solution is an aqueous phase, and the aqueous phase can be a buffered or non-buffered solution; in an optional embodiment, the solvent of the second solution is PBS.

[0063] In an optional embodiment, after the dropwise addition is completed, the mixed solution is continued to be stirred for 3 to 5 hours, preferably 4 hours.

[0064] In an optional embodiment, dialysis is used to remove the solvent of the first solution and the solvent of the second solution in the mixed solution.

[0065] In an optional embodiment, the lipid component includes stearoyl phosphatidylethanolamine-polyethylene glycol, preferably includes stearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0066] In an optional embodiment, the feed 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, preferably 1:5.

[0067] In a specific embodiment, the preparation method of the pharmaceutical composition is as follows: (a) Weigh 5 mg of hypericin and dissolve it in 500 μl of DMSO. Mix well to obtain solution A.

[0068] (b) Weigh 25 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 and dissolve it in 2.5 ml of PBS. Mix well to obtain solution B.

[0069] (c) Solution A was slowly and evenly added dropwise to solution B while stirring continuously, and the reaction was continued by stirring for 4 hours.

[0070] (d) The mixed solution was collected into a dialysis bag and dialyzed against PBS solution for 48 hours to remove DMSO and distearoylphosphatidylethanolamine-polyethylene glycol 2000 that did not form liposomes.

[0071] (e) The dialysate was filtered through a membrane and the volume was fixed to 5 ml with PBS to obtain the desired hypericin-liposome solution stock solution with a concentration of 1 mg / ml.

[0072] In an optional embodiment, the hypericin in any one of the first, second, third and fourth aspects is the hypericin represented by the molecular formula (i), with a CAS number of 548-04-9.

[0073] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0074] The hypericin in Examples 1 to 5 is the hypericin represented by the molecular formula (i), and its CAS number is 548-04-9.

[0075] Example 1 Molecular docking and computer virtual screening of key target proteins in the TGFβ signaling pathway, a key pathway for the onset of pathological scars, were performed. Small molecule natural products were screened from the library (Table 1). Molecular docking showed that hypericin had a lower binding energy with TGFBR1, indicating that hypericin could effectively bind to TGFBR1.

[0076] Table 1 Molecular docking results targeting TGFBR1

[0077] Example 2 Take 10 mg of accurately weighed hypericin powder, dissolve it in 1.9824 ml of DMSO, and stir it thoroughly until the solution is clear to obtain a 10 mM hypericin-DMSO mother solution.

[0078] The above-mentioned hypericin-DMSO stock solution was added to the culture medium of normal skin fibroblasts stimulated by TGFβ according to the dose, so that the final concentration of hypericin in the culture medium was 0, 2.5, 5, 10, and 20 μM, respectively. After 48 hours, the cell proteins were collected, and the expression of type I collagen and type III collagen related to the pathogenesis of pathological scars, as well as the expression of p-SMAD2, an indicator related to the activation of the TGFβ pathway, were detected by Western Blot experiments. The results showed that the expression of type I collagen and type III collagen and the expression of p-SMAD2 in fibroblasts were significantly enhanced after TGFβ stimulation, while their expression was significantly reduced after hypericin treatment, and the degree of reduction was positively correlated with the concentration of hypericin ( Figure 1 ), the above experimental results show that hypericin can inhibit the expression of type I collagen, type III collagen and p-SMAD2.

[0079] Conclusion: The above cell experiments showed that hypericin can down-regulate collagen expression in fibroblasts by inhibiting TGFβ.

[0080] Example 3 Normal fibroblasts were resuspended in serum-free culture medium and added to the upper chamber of the transwell chamber. The total number of cells was 500 / well and the volume was 600 μl. At the same time, the above-mentioned hypericin-DMSO stock solution was added to the upper chamber culture medium according to the dosage, so that the final concentration of hypericin in the culture medium was 0, 2, and 4 μM, respectively, and TGFβ was added for stimulation. 800 μl of normal culture medium was added to the lower chamber of the transwell chamber. After 24 hours, the chamber was removed, the cells in the upper chamber were wiped off, and the cells in the lower chamber were stained with crystal violet. The cells were photographed and the number of cells in each field of view was counted. The number of cells was the number of cells that passed through the transwell membrane in the field of view. The results are as follows: Figure 2 and Figure 3 As shown, the results showed that the migration ability of fibroblasts was enhanced after TGFβ stimulation, while their migration ability was significantly reduced after hypericin treatment.

[0081] Normal skin fibroblasts were cultured to a confluence of 80-90%. A scratch was made on the cell monolayer using a sterile pipette tip or a scratching tool, and floating cells were removed. The cells were cultured using medium containing 0, 2, and 4 μM hypericin, respectively, and stimulated with TGFβ. Photos were taken at 0, 24, and 48 h. The results are shown in Figure 2. Figure 4 As shown, it can be seen that hypericin can effectively inhibit the migration of skin fibroblasts stimulated by TGFβ.

[0082] Conclusion: The above cell experiments showed that hypericin can inhibit TGFβ-induced fibroblast migration.

[0083] Example 4 The hypericin-DMSO mother solution of Example 2 was added to the culture medium of two primary cultured fibroblasts of pathological scar keloid (named KF1 and KF2, respectively) according to the dosage, so that the final concentration of hypericin in the KF1 culture medium was 0, 2.5, 5, 10, 20 μM, and the final concentration in the KF2 culture medium was 0, 2, 4, 8 and 16 μM, respectively. After 48 hours, the cell viability was detected by CCK8 experiment (compared with the group with hypericin concentration of 0), and a dose-effect curve was drawn. The results show that with the increase of hypericin concentration, the viability of keloid fibroblasts decreased significantly ( Figure 5 and Figure 6 ), the half inhibitory concentration (IC50) of hypericin on KF1 and KF2 were 10.33μM and 10.28μM, respectively, indicating that hypericin can inhibit the proliferation of pathological scar fibroblasts.

[0084] Conclusion: The above cell experiments showed that hypericin can inhibit the proliferation of primary cultured fibroblasts of pathological scars.

[0085] Example 5 Preparation of hypericin-DMSO stock solution: Take 10 mg of accurately weighed hypericin powder, dissolve it in 10 ml of DMSO, stir and dissolve until the solution is clear, and obtain 1 mg / ml hypericin-DMSO stock solution.

[0086] Preparation of hypericin-liposome solution mother solution: accurately weigh 10 mg of hypericin powder and dissolve it in 1 ml of DMSO, mix well to obtain solution A. Weigh 50 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and dissolve it in 5 ml of PBS, mix well to obtain solution B. Slowly and evenly add solution A to solution B, stirring constantly while adding, and then continue to stir and react 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 has not formed liposomes. Filter the above dialysate through a membrane and dilute it to 10 ml with PBS to obtain the required hypericin-liposome solution mother solution with a concentration of 1 mg / ml (based on hypericin content).

[0087] The hyperforin solutions prepared by two methods were used for animal experiments. The experimental scheme was as follows: the hyperforin-DMSO stock solution and the hyperforin-liposome stock solution were diluted to 50 μg / ml with PBS. Hypertrophic scar modeling was performed on the rabbit ears. The modeling method was as follows: the rabbit was fixed in a fixator, the head was exposed, and the hair was removed with a hair shaver. The skin was disinfected with alcohol cotton balls, propofol 5 mg / kg was slowly injected into the ear vein or isoflurane was continuously inhaled for anesthesia. After successful anesthesia, a skin trephine was used to drill circular wounds at the selected sites. Each wound was 1.5-2 cm apart and the depth reached the surface of the cartilage. The skin was removed with tweezers and scissors, and the perichondrium was removed with a blade and scissors, and the cartilage was retained. Erythromycin ointment was given after surgery to prevent wound infection. Bleeding and secretion were observed within 3 days after surgery, and then the rabbits were allowed to drink water and eat freely, and the wounds were allowed to heal naturally. The modeling was considered successful when scar tissue that was significantly higher than normal skin and did not exceed the scope of the wound surface was formed at the wound. On the third day after modeling, the rabbits were divided into three groups and 100 μl of 50 μg / ml hypericin-DMSO solution, 50 μg / ml hypericin-liposome solution or an equal amount of PBS solution were applied to the wounds every day. Scar formation was recorded on different days after medication, and the size and color of the scar were analyzed on the 30th day after modeling.

[0088] The experimental results are compared as follows: On the 30th day of medication, the hyperforin + liposome group had the best effect among the three groups. The hypertrophic scar formed was the smallest and the scar color was the lightest, close to the color of the normal skin next to it. Its therapeutic effect was much better than that of the hyperforin + DMSO group. The PBS group had the worst effect. The hypertrophic scar area was large and the scar color was red ( Figure 7 and Figure 8 ).

[0089] Conclusion: The results of the above comparative experiments show that the treatment effect of hyperforin + liposome group on hypertrophic scars is significantly better than that of hyperforin + DMSO group. Liposome increases the number of hyperforin penetrating the skin and entering cells, and avoids the toxic side effects caused by DMSO.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of hypericin in one or more of (I) to (IV): (I) preparing a drug for treating and / or preventing pathological scars in a subject; (II) preparing a medicament for treating and / or preventing non-pathological scars in a subject; (III) preparing a drug for downregulating at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in a subject; (IV) Downregulation of at least one of type I collagen, type III collagen and phosphorylated SMAD2 protein in subjects for non-diagnostic and non-therapeutic purposes.

2. The use according to claim 1, characterized in that: 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 use according to claim 1 or 2, characterized in that: In said application, hypericin is delivered by a delivery formulation.

4. A method 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, characterized in that: Comprising administering hypericin to a subject.

5. The method according to claim 4, characterized in that The subject comprises fibroblasts.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition 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.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition contains a delivery preparation for delivering hypericin; and / or the pharmaceutical composition is an external preparation.

8. The pharmaceutical composition according to claim 7, characterized in that The delivery formulation includes liposomes, nanoparticles, proteins, polypeptides, cationic polymers, nucleic acid aptamers or small molecule compounds.

9. The method for preparing the pharmaceutical composition according to any one of claims 6 to 8, characterized in that: The method comprises mixing hypericin with pharmaceutically acceptable auxiliary materials.

10. The preparation method according to claim 9, characterized in that: The method comprises the steps of dropping a first solution into a second solution, stirring the second solution during the dropping process; continuing to stir the mixed solution after the dropping process is completed; and then removing the solvent of the first solution and the solvent of the second solution from the mixed solution; The first solution contains hypericin; the second solution contains a lipid component for constituting liposomes.

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