Medicine and application of umbilical cord-derived mesenchymal stem cell exosome in preparation of medicine for treating and / or preventing anorectal diseases

Abstract

The invention provides a medicine and application of an umbilical cord-derived mesenchymal stem cell exosome in preparation of a medicine for treating and / or preventing anorectal diseases, and relates to the technical field of medicine application. The exosome is a subcellular component secreted by living cells, contains cell specific protein, lipid and nucleic acid, can be used as a signal molecule to be transmitted to other cells so as to change the functions of other cells, and has the effects of sterilization (drug-resistant bacteria), inflammation resistance, virus resistance and immune regulation. The exosome is free of anaphylactic reaction, toxic and side effects and irritation and can be used for a long time. The application of the traditional Chinese medicine composition in prevention and treatment of haemorrhoids is found for the first time, and an effective choice without toxic and side effects is provided for patients with haemorrhoids.

Description

Technical Field

[0001] The present invention relates to the technical field of drug application, and in particular to application of a drug and umbilical cord-derived mesenchymal stem cell exosomes in the preparation of drugs for treating and / or preventing anorectal diseases. Background Art

[0002] Hemorrhoids are a common chronic disease of the anal organs at the end of the digestive system, in the category of metabolic chronic diseases. Hemorrhoids refer to the soft venous masses formed by the expansion, bending, bulging and swelling of the anal canal wall and the blood vessels inside and around the lower rectum under the mucosa at the end of the human body. According to the location of the hemorrhoids, they can be divided into external hemorrhoids, internal hemorrhoids and mixed hemorrhoids. External hemorrhoids (external) are formed near the anus, hidden under the sensitive skin, and usually do not cause pain, but blood clots form under the skin around the anus, which can cause obvious pain. Internal hemorrhoids (internal) are formed inside the anus, under the mucosa, and common symptoms are bleeding and protrusion during defecation. Mixed hemorrhoids are a mixture of internal and external hemorrhoids.

[0003] The pathogenesis of hemorrhoids is mainly related to abnormal immune cell function and anal mucosal damage, and inflammation causes vascular oxidative damage. Hemorrhoids are mainly manifested clinically as: blood in stool, prolapse, anal swelling, pain, foreign body sensation in anus, etc. At present, the treatment of hemorrhoids is divided into surgical treatment and non-surgical treatment, and surgical treatment is mainly applicable to III and IV stage internal hemorrhoids. Non-surgical treatment is divided into oral medication, topical medication, injection therapy and ligation therapy, other physical therapies. Due to the special hemorrhoidal lesion site, the effect of taking medicine is not good, and the clinical existing hemorrhoidal topical medicine treatment effect is also not ideal, and surgical treatment is easy to relapse, and is accompanied by side effects. Therefore, researchers in this field are always exploring the medicine for effectively preventing and treating hemorrhoids.

[0004] For example, Chinese patent CN111840389A discloses a medicine for treating hemorrhoids, which includes the following components in parts by weight: 8-15 parts of Herba Cynomorii, 10-20 parts of Salvia miltiorrhiza, 6-10 parts of Angelica sinensis, 10-30 parts of Astragalus, 8-10 parts of Frosted Morus alba, 8-12 parts of Pheretima, 10-15 parts of Myrrha, 10-15 parts of Rhizoma Chuanxiong, 10-30 parts of Double Flower, and 10-30 parts of Adzuki Bean. It is prepared by using pure natural plant Chinese herbal medicine, but it takes three to four courses of treatment to be effective, and the treatment effect is not ideal, which cannot meet the needs of patients. Chinese patent CN111840464A discloses a preparation for treating hemorrhoids and its preparation method. The preparation is prepared by using Lithospermum officinale, Sanguisorba officinalis, Coptis chinensis, Sichuan pepper and Scutellaria baicalensis as main drugs; using Herba Lycopodii, Herba Menthae, Herba Agrimoniae and Cortex Dictamni as assistant drugs, and is used for removing dampness and swelling, treating sores, boils, swelling and poison, astringing and stopping bleeding and tonifying deficiency; using Sophora japonica, Sophora japonica, Pomegranate peel, Sophora flavescens, Rehmannia root, Angelica dahurica, Bletilla striata, Cortex Phellodendri, Polygonum cuspidatum, calcined gypsum, pearl powder, silver beads, alum, Sophora japonica salt, L-tetrahydropalmatine and Borneol as adjuvant drugs. However, the preparation is complicated in composition, cumbersome in preparation and slow in effect.

[0005] Exosomes are a type of extracellular vesicles with a diameter of 50-150nm, which are secreted by cells under physiological or pathological conditions. Exosomes have a double membrane structure, and the main components include proteins, RNA, DNA, amino acids and various metabolites. Studies have shown that nucleic acids, proteins and other components in exosomes have the characteristics of transmitting genetic information and regulating physiological functions. Exosomes from different sources have different functions. Among them, milk exosomes, as one of the important components of milk, can carry and transmit signal molecules such as miRNA, and have become a hot topic in the current research of food-derived exosomes. A study compared and analyzed the milk exosome miRNA of cattle, pigs, humans and pandas, and found that the miRNA enriched in milk exosomes are related to events such as enhancing immunity, regulating metabolism, and resisting diseases.

[0006] Exosomes, as a subcellular component secreted by living cells, have bactericidal (resistant bacteria), anti-inflammatory, antiviral and immune-modulating effects. Exosomes not only have no allergic reactions, no toxic side effects, no irritation, and can be used for a long time. The prior art has disclosed some applications of exosomes, such as Chinese patent CN 116867479A discloses a skin improvement cosmetic composition containing black yeast-derived exosomes as an effective ingredient, which can be used to improve skin elasticity, improve skin wrinkles, improve skin texture, improve skin color, improve skin brightness, regenerate skin, moisturize skin and / or whiten. Chinese patent CN116473919A discloses an exosome eye drop for treating optic nerve damage and a preparation method thereof, which has the effects of inhibiting inflammatory reactions, protecting nerve cells, and alleviating retinal damage caused by various reasons.

[0007] At present, there are no reports on the research of exosomes in the treatment of hemorrhoids. Summary of the invention

[0008] In view of the above problems, the present invention provides an application of umbilical cord-derived mesenchymal stem cell exosomes in hemorrhoid medicine.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] On the one hand, the present invention provides a use of umbilical cord-derived mesenchymal stem cell exosomes in the preparation of a drug for treating and / or preventing anorectal diseases.

[0011] Preferably, the drug comprises umbilical cord-derived mesenchymal stem cell exosomes.

[0012] Preferably, the umbilical cord-derived mesenchymal stem cell exosomes are a lyophilized preparation.

[0013] Preferably, the drug is in a pharmaceutically acceptable external dosage form.

[0014] Further preferably, the external dosage form also includes common pharmaceutical excipients and is prepared into dry powder, water-soluble agent, oily agent, ointment or suppository.

[0015] Preferably, the drug is in a pharmaceutically acceptable oral dosage form.

[0016] Preferably, the drug is in a pharmaceutically acceptable injection form.

[0017] Preferably, the drug may further comprise a pharmaceutically acceptable carrier.

[0018] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0019] Preferably, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginic acid, gelatin, gum arabic, glyceryl monostearate, sodium starch glycolate, guar gum, glycerol, and propylene glycol.

[0020] Preferably, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0021] Preferably, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglyceryl-3 polyricinoleate.

[0022] Preferably, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan.

[0023] Preferably, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0024] Preferably, the binder is selected from at least one of ethanol, starch slurry, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinyl pyrrolidone, gum arabic, gelatin, and alginic acid.

[0025] Preferably, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0026] Preferably, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, and poloxamer.

[0027] Preferably, the pH adjuster is at least one selected from citric acid, fumaric acid, succinic acid, tartaric acid, malic acid and ascorbic acid.

[0028] Preferably, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide or formamide.

[0029] Preferably, the flavoring agent is selected from at least one of sweet orange flavor, vanilla flavor, strawberry flavor, milk flavor, banana flavor, and cherry flavor.

[0030] Preferably, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate and sodium bicarbonate.

[0031] Preferably, the anorectal disease comprises hemorrhoids, fistulas, fissures, functional bowel disorders, colorectal inflammatory diseases and colorectal tumors.

[0032] Further preferably, the anorectal diseases include hemorrhoids, anal fissures, anal fistulas, perianal abscesses, anal skin diseases, anal sinusitis, proctitis, rectal ulcers, outlet constipation, rectal prolapse, rectocele, rectal mucosal prolapse, anorectal stenosis, anal incontinence, anal canal cancer, rectal cancer, anal papilloma, rectal polyps, anorectal tuberculosis, anal neurosis, condyloma acuminatum, anorectal congenital malformations and anorectal trauma.

[0033] Further preferably, the anorectal disease is hemorrhoids.

[0034] More preferably, the hemorrhoids include internal hemorrhoids, external hemorrhoids and mixed hemorrhoids.

[0035] Preferably, the anorectal disease is accompanied by pain, edema, itching, prolapse and / or blood in the stool.

[0036] In another aspect, the present invention provides a method for preparing a drug for treating and / or preventing anorectal diseases, wherein the drug comprises a therapeutically effective amount of umbilical cord-derived mesenchymal stem cell exosomes.

[0037] Preferably, the drug is in a pharmaceutically acceptable external dosage form.

[0038] Preferably, the drug is in a pharmaceutically acceptable oral dosage form.

[0039] Preferably, the drug is in a pharmaceutically acceptable injection form.

[0040] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0041] Preferably, the medicament further comprises active substances required for treating anorectal diseases.

[0042] Preferably, the drug inhibits the up-regulation of the expression of inflammatory factors.

[0043] More preferably, the inflammatory factors include IL-6, IL-1β and TNF-α.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] Exosomes, as subcellular structures secreted by living cells, have a variety of active ingredients, and have bactericidal (resistant bacteria), anti-inflammatory, antiviral and immune-modulating effects. Exosomes not only have no allergic reactions, no toxic side effects, no irritation, and can be used for a long time. The present invention has discovered its application in the prevention and treatment of hemorrhoids for the first time. Each dose group of the exosome preparation of the present invention has a therapeutic effect on the rat hemorrhoid model induced by glacial acetic acid. Its mechanism of action may be related to local tissue antibacterial, anti-inflammatory and reduction of the release of inflammatory factors such as IL-6, IL-1β, TNF-α, and reduction of anal tissue pathological damage. It provides an effective and non-toxic option for patients with hemorrhoids. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0047] Test Case

[0048] Experimental study on the pharmacodynamic evaluation and mechanism analysis of umbilical cord-derived mesenchymal stem cell exosomes in the treatment of hemorrhoids

[0049] 1. Experimental Materials

[0050] 1. Experimental Animals

[0051] 56 SPF male SD rats, weighing (178±20) g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0052] 2. Experimental reagents and drugs

[0053] Glacial acetic acid: mass fraction ≥99.9%, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number A226173;

[0054] Rat interleukin-6 (IL-6) kit: (lot #ERC005), purchased from Xinbosheng Biotechnology Co., Ltd.;

[0055] IL-1β kit: (lot #ERC009), purchased from Xinbosheng Biotechnology Co., Ltd.;

[0056] Tumor necrosis factor-α (TNF-α) kit: (lot #ERC103a), purchased from Xinbosheng Biotechnology Co., Ltd.;

[0057] Positive drug: Mayinglong Musk Hemorrhoid Ointment (Mayinglong Pharmaceutical Group Co., Ltd., batch number 191203).

[0058] Exosomes: Exosome freeze-dried preparation (batch number 20241022), obtained from the culture supernatant of umbilical cord-derived mesenchymal stem cells, was isolated and purified and provided by Beijing Regeneration Century Biotechnology Co., Ltd. Reconstitute before use and spray on the affected area.

[0059] 3. Main instruments

[0060] 1 / 10 000 precision balance: Mettler-Toledo Instruments Shanghai Co., Ltd., model ME204E;

[0061] Low-temperature high-speed centrifuge: Eppendorf, model 5425R;

[0062] Enzyme-linked immunosorbent assay: BioTek, USA, model SYNERGYH1.

[0063] 2. Experimental Methods

[0064] 1. Animal Modeling and Drug Administration

[0065] Acute hemorrhoids model was prepared with glacial acetic acid, and rats were randomly divided into normal control group, model group, blank control group (only excipients, no exosomes), Mayinglong Musk Hemorrhoids Ointment (positive drug, specification 20g / tube, dose 1g / kg) group, and exosome preparation group (divided into 3 groups; low dose: 10 billion particles / mL, medium dose: 20 billion particles / mL, high dose: 50 billion particles / mL), 8 rats in each group. The control group was not treated with anything.

[0066] Acute hemorrhoid model prepared with glacial acetic acid: unfold filter paper pieces of equal size and inner diameter of 6mm and soak them in glacial acetic acid. Place the filter paper pieces soaked with glacial acetic acid close to the perianal skin and mucosa for 0.5min, use one piece of filter paper each time, and change it twice. This will cause rectal mucosal edema and induce a typical inflammatory reaction.

[0067] Drug administration after modeling: In the exosome preparation group, the spray head was directly pushed into the rat anus, and the exosome preparation and Mayinglong Musk Hemorrhoids Ointment were 2 cm inside the anus. The exosome group was given the drug twice a day for 3 days, for a total of 6 times; the Mayinglong Musk Hemorrhoids Ointment was given twice a day for 3 days, for a total of 6 times.

[0068] 2. Detection of serum inflammatory factors

[0069] Whole blood was collected via the abdominal aorta into ordinary blood collection tubes, placed at room temperature for 2 hours, and then centrifuged at 4°C and 3000 r / min for 15 minutes to separate the serum, which was packaged and frozen at -80°C for the detection of serum IL-6, IL-1β, and TNF-α levels. The specific operation was carried out strictly in accordance with the instructions of the ELISA kit.

[0070] 3. Detection of inflammatory factors in anal tissue

[0071] The rectal tissue from the anus to the rectum of each group of rats was cut and homogenized using a tissue grinder. The tissue was centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant was collected and packaged and frozen at -80°C for detection of IL-6, IL-1β, and TNF-α inflammatory factor levels. The specific operation was carried out strictly in accordance with the instructions of the ELISA kit.

[0072] 4. Gross anatomy of tissues surrounding the anus

[0073] The rectal tissue from the anus to the rectum was cut from each group of rats, and the gross findings of the tissue were observed under a gross dissection microscope, including whether there was redness, swelling, tissue defect, exudate, and bleeding spots.

[0074] 5. Statistics

[0075] Graphpad Prism 8 software was used, and the results of various indicators were The t-test was used to compare the means between two groups, and one-way ANOVA and pairwise comparison (LSD method) were used to compare the measurement data among multiple groups.

[0076] 3. Experimental Results

[0077] 1. Effects of exosome preparations on rats with acute hemorrhoids induced by glacial acetic acid

[0078] Effect of glacial acetic acid on rat body weight Compared with the normal control group, the body weight of the model group rats was significantly reduced on the third day after modeling (P < 0.05). Compared with the model group, the exosomes dose groups and the Mayinglong Musk Hemorrhoids Ointment group had an improvement effect on the weight loss of rats, and the difference in the exosome preparation 3g group was significant on the third day (P < 0.05). The results are shown in Table 1.

[0079] Table 1. Effects of exosome preparations on body weight in rat hemorrhoid model induced by glacial acetic acid ( n=8)

[0080]

[0081] Compared with the control group: #P<0.05; compared with the acetic acid model group: *P<0.05

[0082] Effects on serum IL-6, IL-1β and TNF-α levels Compared with the normal control group, the serum IL-6, IL-1β and TNF-α levels of rats in the model group were significantly increased (all P less than 0.05). Compared with the model group, the serum IL-6, IL-1β and TNF-α levels of rats in the exosome preparation groups and the Mayinglong Musk Hemorrhoids Ointment group were significantly decreased (all P less than 0.05). The results are shown in Table 2.

[0083] Table 2. Effects of exosome preparations on serum IL-6, IL-1β and TNF-α levels in rat hemorrhoids model induced by acetic acid ( n=8)

[0084]

[0085]

[0086] Compared with the normal control group: ##P<0.05, ##P<0.01, ###P<0.001; compared with the glacial acetic acid model group: ***P<0.05; **P<0.01; ***P<0.001.

[0087] Effects on the levels of IL-6, IL-1β and TNF-α in anal tissue Compared with the normal control group, the levels of IL-6, IL-1β and TNF-α in anal tissue of rats in the model group were significantly increased (all P less than 0.05). Compared with the model group, the levels of IL-6, IL-1β and TNF-α in anal tissue of rats in each dose group of exosome preparation and Mayinglong Musk Hemorrhoids Ointment group were significantly decreased (all P less than 0.05), the results are shown in Table 3.

[0088] Table 3. Effects of exosome preparations on the levels of IL-6, IL-1β and TNF-α in anal tissue of rat hemorrhoids model induced by acetic acid ( n=6)

[0089]

[0090] Compared with the normal control group: #P<0.05, ##P<0.01; compared with the acetic acid model group: *P<0.05, **P<0.01, ***P<0.001.

[0091] Anal and rectal tissue:

[0092] There was no abnormality in the anal tissue of the rats in the normal control group. The anal mucosal tissue of the rats in the model group was necrotic, swollen, exuded, and had bleeding spots. The anal and rectal tissue damage of the rats in the exosomes dose groups and the Mayinglong Musk Hemorrhoids Ointment group was significantly improved, with no obvious redness and swelling, mucosal tissue bleeding was reduced, and the boundaries were relatively clear.

[0093] Each dose group of the exosome preparation provided by the present invention has a therapeutic effect on the rat hemorrhoid model induced by glacial acetic acid. Its mechanism of action may be related to local tissue antibacterial, anti-inflammatory and reduction of the release of inflammatory factors such as IL-6, IL-1β, and TNF-α, and alleviation of anal tissue pathological damage.

[0094] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of umbilical cord-derived mesenchymal stem cell exosomes in the preparation of drugs for treating and / or preventing anorectal diseases.

2. The use according to claim 1, characterized in that: The drug comprises umbilical cord-derived mesenchymal stem cell exosomes and / or a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that: The drug is in a pharmaceutically acceptable external dosage form, oral dosage form or injection dosage form.

4. The use according to claim 3, characterized in that: The external dosage form also includes pharmaceutically acceptable auxiliary materials and is prepared into dry powder, water-soluble agent, oily agent, ointment or suppository.

5. The use according to claim 1, characterized in that: The anorectal diseases include hemorrhoids, fistulas, fissures, functional bowel disorders, colorectal inflammatory diseases and colorectal tumors.

6. The use according to claim 5, characterized in that: The anorectal diseases include hemorrhoids, anal fissures, anal fistulas, perianal abscesses, anal skin diseases, anal sinusitis, proctitis, rectal ulcers, outlet constipation, rectal prolapse, rectocele, rectal mucosal prolapse, anorectal stenosis, anal incontinence, anal canal cancer, rectal cancer, anal papilloma, rectal polyps, anorectal tuberculosis, anal neurosis, condyloma acuminatum, anorectal congenital malformations and anorectal trauma.

7. The use according to claim 5, characterized in that: The hemorrhoids include internal hemorrhoids, external hemorrhoids and mixed hemorrhoids.

8. A drug for treating or preventing anorectal diseases, comprising a therapeutically effective amount of umbilical cord-derived mesenchymal stem cell exosomes.

9. The drug according to claim 8, characterized in that The medicine further comprises a pharmaceutically acceptable carrier and / or an active substance required for treating anorectal diseases.

10. The drug according to claim 8, characterized in that The drug inhibits the up-regulation of the expression of inflammatory factors.

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