Application of glycyrrhizic acid in preparation of medicine for preventing or treating prostatitis

Abstract

The invention provides application of glycyrrhizic acid in preparation of a medicine for preventing or treating prostatitis. Experiments show that glycyrrhizic acid can significantly relieve inflammatory response and pain symptoms in prostate tissues; meanwhile, polarization of M1 type macrophages can be effectively inhibited, so that inflammatory response of chronic abacterial prostatitis is relieved, and the compound can be used for developing related drugs for treating chronic abacterial prostatitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, relates to a new medicinal use of glycyrrhizic acid, and specifically relates to the use of glycyrrhizic acid in preparing a medicine for preventing or treating prostatitis. Background Art

[0002] Prostatitis is one of the most common diseases in urology practice. According to epidemiological data, the prevalence of prostatitis symptoms ranges from 2.2% to 9.7%, with an average incidence of 8.2% (Krieger JN. Epidemiology of prostatitis. Int J Antimicrob Agents 2008; 31 Suppl 1 (Suppl 1): S85-90.). According to the classification criteria established by the National Institutes of Health (NIH), prostatitis is divided into four categories. Among them, type III chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) is the main form of prostatitis, accounting for more than 90% of all cases (Krieger JN. NIH consensus definition and classification of prostatitis. Jama 1999; 282 (3): 236-237.). The disease presents with a variety of symptoms, including persistent pelvic discomfort, dysuria, sexual dysfunction, fatigue, and memory loss, which seriously affect the patient's health and quality of life (Clemens JQ. Urologic chronic pelvic pain syndrome: insights from the MAPP Research Network. Nat Rev Urol 2019; 16(3): 187-200.). Due to the complex pathogenesis of CP / CPPS and the limited understanding of it, there are still few effective treatment strategies. There is an urgent need to develop new therapeutic drugs. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides the use of glycyrrhizic acid in the preparation of a drug for preventing or treating prostatitis, especially a drug for chronic non-bacterial prostatitis. Glycyrrhizic acid is a triterpenoid saponin, an active ingredient in licorice, which can antagonize high mobility group protein B1 (HMGB1) and has multiple pharmacological effects such as anti-inflammatory, antiviral and immunomodulatory. Currently, there is no report on the use of glycyrrhizic acid in the treatment of chronic non-bacterial prostatitis.

[0004] The present invention adopts the following technical solution:

[0005] Application of glycyrrhizic acid in the preparation of a drug for preventing or treating prostatitis, wherein the glycyrrhizic acid has a molecular formula of C 42 H62 O 16 , the chemical structure is:

[0006]

[0007] According to one embodiment of the present invention, the application is the application of glycyrrhizic acid in the preparation of a drug for preventing or treating chronic nonbacterial prostatitis. Further, the application is to alleviate the inflammatory response and pain symptoms caused by chronic nonbacterial prostatitis.

[0008] According to one embodiment of the present invention, the application is a drug that can be used clinically and is prepared using glycyrrhizic acid as an active ingredient using conventional preparation processes.

[0009] According to one embodiment of the present invention, the application is a drug that can be used clinically and is prepared by conventional preparation technology with glycyrrhizic acid as the only active ingredient or with glycyrrhizic acid as the main active ingredient in combination with other drugs.

[0010] According to one embodiment of the present invention, the drug for treating chronic non-bacterial prostatitis of the present invention further comprises a pharmaceutically acceptable carrier, including but not limited to: a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent or an adsorption carrier.

[0011] According to one embodiment of the present invention, the drug can be prepared into, but not limited to, injection, tablets, powders, granules, and capsules. The above-mentioned various dosage forms of the drug can be prepared according to conventional methods in the pharmaceutical field. The drug can be administered alone; or in combination with other drugs capable of treating chronic non-bacterial prostatitis.

[0012] The "effective amount" used in the present invention refers to an amount that can produce functions or activities on humans and / or animals and can be accepted by humans and / or animals.

[0013] The effective amount of the glycyrrhizic acid of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the glycyrrhizic acid such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc.

[0014] Beneficial effects:

[0015] The present invention provides the use of glycyrrhizic acid in the preparation of a drug for preventing or treating prostatitis. Experiments have found that glycyrrhizic acid can significantly reduce the inflammatory response and pain symptoms in prostate tissue; at the same time, it can effectively inhibit the polarization of M1 macrophages, thereby reducing the inflammatory response of chronic non-bacterial prostatitis, and can be used to develop related drugs for treating chronic non-bacterial prostatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a diagram of behavioral experiment results, showing that glycyrrhizic acid alleviates the response to pain stimulation in mice with experimental autoimmune prostatitis (EAP);

[0017] Figure 2 This is a diagram showing the high expression of HMGB1 protein in the prostate tissue of mice with experimental autoimmune prostatitis;

[0018] Figure 3 This is a picture of hematoxylin-eosin staining results, showing the inhibitory effect of glycyrrhizic acid on inflammatory cell infiltration in mice with experimental autoimmune prostatitis;

[0019] Figure 4 It is an enzyme-linked immunosorbent assay (ELISA) experimental result diagram, which shows that glycyrrhizic acid reduces the inflammatory factors IL6 and TNF-α in the serum of mice with experimental autoimmune prostatitis;

[0020] Figure 5 The figure is the result of iNOS immunohistochemistry, which shows that glycyrrhizic acid reduces the number of M1 macrophages in the prostate tissue of mice with experimental autoimmune prostatitis. DETAILED DESCRIPTION

[0021] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further illustrated by specific embodiments below. It should be understood that these embodiments are only helpful for understanding the present invention and should not be regarded as specific limitations of the present invention. Unless otherwise specified, the parts described in the present invention are all parts by weight, and the percentages are all mass percentages.

[0022] The raw materials and reagents used in the present invention are all commercially available products, wherein: NOD mice were purchased from Jiangsu Jicui Yaokang Biological Co., Ltd.; SD rats were purchased from Chongqing Enbi Biotechnology Co., Ltd.; RAW264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); glycyrrhizic acid was purchased from MedChem Express with item number: HY-N0184, and glycyrrhizic acid was first prepared into a stock solution in 100% DMSO, and then diluted to a working concentration with a mixed solution containing 5% DMSO, 40% PEG300, 5% Tween 80 and 50% sterile water.

[0023] Example 1

[0024] Prostate protein antigens were prepared using Sprague-Dawley rats. After euthanasia of the rats, the prostate tissues were removed, washed, and homogenized in a solution containing 0.5% TritonX100 and protease inhibitors. The supernatant was emulsified with complete Freund's adjuvant at a ratio of 1:1 to form a stable "water-in-oil" emulsion. This antigen emulsion was used for immunization at multiple subcutaneous sites (bilateral inguinal regions, shoulders, and tail base) of NOD mice, with a total injection volume of 150 microliters per mouse. Immunizations were performed on days 0 and 28, respectively, and the control group received PBS injections at the same sites.

[0025] On the second day after the second immunization, mice began to receive intraperitoneal injections for 14 consecutive days. The drug was glycyrrhizic acid at a dose of 5 mg / kg. The preparation method of glycyrrhizic acid was as follows: first, it was dissolved in 100% DMSO to prepare a stock solution, and then diluted with a mixed solution containing 5% DMSO, 40% PEG300, 5% Tween 80 and 50% sterile water to obtain the required working concentration.

[0026] On day 42 after induction, mice underwent behavioral testing and were subsequently euthanized to collect blood and prostate tissue for subsequent analysis.

[0027] Behavioral testing:

[0028] Before the mice were killed, the Von Frey mechanical prick test kit was used to evaluate the responsiveness of the mice to painful stimulation. The stimulation was limited to the lower abdominal area near the prostate, and attention was paid to different places within the stimulation area to avoid desensitization effects. The specific operation was as follows: the mice were placed in a separate plastic room with a stainless steel mesh floor and allowed to adapt to the new environment for 30 minutes. Subsequently, 5 separate Von Frey fibers (0.04, 0.16, 0.4, 1.0 and 4.0 g) were used to stimulate the mice separately, and the responsiveness of each mouse to the stimulation was tested. Each stimulation lasted for 1 to 2 seconds, with an interval of 5 seconds between each stimulation, for a total of 10 times. Three types of behavior were identified as positive responses to stimulation: (1) sharp contraction of the abdomen; (2) immediate licking or scratching of the area stimulated by the fiber; (3) jumping. The results are expressed as the percentage of positive reactions.

[0029] The results are as follows Figure 1 As shown: The responsiveness of mice in the glycyrrhizic acid treatment group to pain stimulation was significantly weakened, indicating that glycyrrhizic acid alleviates the pain symptoms caused by prostatitis.

[0030] Example 2

[0031] The expression of HMGB1 in the prostate tissue of mice with experimental autoimmune prostatitis was investigated by Western blot.

[0032] (1) Take an appropriate amount of frozen prostate tissue and cut it into pieces. Add 250ul RIPA lysis buffer to every 20mg of tissue.

[0033] (2) Mix the protein phosphatase inhibitor cocktail with tissue cell lysate at a ratio of 1:100.

[0034] (3) The tissue was further crushed using a tissue homogenizer and lysed on ice for 30 min.

[0035] (4) After the sample is fully lysed, centrifuge at 12000 rpm / min at 4°C for 15 min and collect the supernatant.

[0036] (5) Transfer to a new 1.5 ml EP tube and record the volume; retain 3 ul of protein from each sample for BCA concentration determination, collect 100 ul for the following experimental studies, and store 100 ul at -80 degrees for backup.

[0037] (6) Add 1 / 4 volume of 5× SDS loading buffer to each EP tube, heat at 99°C for 10 min, lyse and cool at -20°C for 20 min, and store at -80°C after aliquoting.

[0038] Add 1 / 4 volume of 5X SDS-PAGE Sample Loading Buffer to the above 1.5 ml EP tube. Mix thoroughly by pipetting. Place the sample in a preheated 100°C water bath and heat for 10 min to fully denature the protein.

[0039] (7) Cool the protein sample on ice and centrifuge it before performing a Western blot experiment or store it in a -80°C refrigerator for a long time for future use.

[0040] (8) Each experiment was repeated three times and the Western blot results were quantitatively analyzed.

[0041] The results are as follows Figure 2 As shown: In the experimental autoimmune prostatitis mouse model, HMGB1 protein showed high expression levels in prostate tissue, which clearly revealed a significant correlation between HMGB1 and prostatitis. Therefore, targeted inhibition of HMGB1 expression is expected to become an effective strategy for the treatment of prostatitis.

[0042] Example 3

[0043] Hematoxylin-eosin staining was used to evaluate the pathological changes of chronic nonbacterial prostatitis treated with glycyrrhizic acid.

[0044] (1) Baking: Place the slices on a rack and bake in a 70°C constant temperature electric oven for 1 h until the paraffin is completely melted.

[0045] (2) Dewaxing: Quickly immerse the slide rack in the following order: xylene I, xylene II for 30 min each, 100% alcohol I, 100% alcohol II, 95% alcohol, 80% alcohol, and 70% alcohol for 3 min each, and then rinse with tap water for 3 min.

[0046] (3) Hematoxylin staining: Soak the dewaxed slice holder completely in the hematoxylin solution for 30 min, and then rinse with tap water until clear and transparent.

[0047] (4) Decolorization and blueing: Place the slice rack completely in 1% hydrochloric acid alcohol for 10 seconds for differentiation, rinse with tap water for 1 minute; then place it in 1% ammonia solution for blueing for 1 minute, and rinse with tap water for 3 minutes.

[0048] (5) Eosin staining: Soak the slice holder completely in eosin solution for 30 min, and rinse with tap water until clear and transparent.

[0049] (6) Dehydration: Immerse the slice rack in 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol I, and 100% alcohol II for 30 seconds each, and then put it in xylene I and xylene II for 10 minutes each.

[0050] (7) Sealing: Dip a cotton swab in neutral gum and drop it on the tissue. Slowly cover the tissue with a coverslip, taking care to avoid creating bubbles. Place the sealed sections in a fume hood to dry.

[0051] The experimental results are as follows Figure 3 As shown: the number of inflammatory infiltrating cells in the glycyrrhizic acid group was significantly reduced, indicating that glycyrrhizic acid has an inhibitory effect on the infiltration of inflammatory cells in mice with experimental autoimmune prostatitis.

[0052] Example 4

[0053] The concentrations of IL-6 and TNF-α in mouse serum were detected using ELISA kits specific for IL-6 (CSB-E04639m) and TNF-α (CSB-E04741m), both of which were purchased from Cusabio. The experiments were carried out in strict accordance with the operating procedures provided by the manufacturer.

[0054] The experimental results are as follows Figure 4 Shown: Glycyrrhizic acid reduced the inflammatory factors IL6 and TNF-α in the serum of mice with experimental autoimmune prostatitis.

[0055] Example 5

[0056] Immunohistochemical staining was used to determine the proportion of M1 polarized macrophages in prostate tissue after glycyrrhizic acid treatment

[0057] (1) Dewaxing and rehydration of sections

[0058] Paraffin sections were placed in a 60°C thermostat for 120 min / 20 min. Dewaxing and hydration: xylene I (30 min) → xylene II (30 min) → anhydrous ethanol (5 min × 2 times) → 95% ethanol (5 min) → 90% (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min). Rinse with PBS for 5 min × 3.

[0059] (2) Antigen retrieval: for formalin-fixed paraffin-embedded tissue microarrays

[0060] Microwave heating: Heat 0.01 mol / L sodium citrate buffer (pH 6.0) in a microwave oven, put the tissue chip in after boiling for 4 minutes, turn off the power, wait for 6 minutes, and heat again for 20 seconds. After cooling to room temperature (2 hours), wash with PBS 3 times × 5 minutes (depending on the condition of the slide, you can refer to the microwave heating "3-5-3 method" of 3 minutes of warm boiling, then let it stand for 5 minutes, and then warm boiling for 3 minutes).

[0061] (3) Elimination of endogenous peroxidase activity

[0062] 20ml 3% H2O2, dilute to 200ml with methanol, soak at room temperature for 15 minutes, wash 3 times with PBS for 5 minutes.

[0063] (4) Closed

[0064] Take out the slide, wipe off the liquid between groups with absorbent paper, draw circles with a PAP pen and add blocking solution, and block for 1.5 hours

[0065] Blocking solution: 0.2g BSA + 10ml PBS + 100ul 1% Triton.

[0066] (5) Primary Antibody Reaction

[0067] Prepare 1% BSA (0.1g BSA + 10ml PBS) antibody diluent; remove the blocking solution in the circle, add 1 drop of 1% BSA to wash, and then remove; add antibodies according to different antibody titers (1:50, 1:100, 1:150, 1:200), incubate at 37℃ for 2-3h, or at 4℃ overnight. Take out and rewarm for 20min; rinse with PBS for 5min×3 times to wash away the antibodies.

[0068] (6) Secondary antibody reaction

[0069] IHC: shake off PBS on the slide, place in a bathtub, add secondary antibody in the secondary antibody kit (not less than 100ul), cover with sealing film, incubate at room temperature for 20 minutes, and wash with PBS for 5 minutes × 4 times.

[0070] (7) Chromogenic (IHC)

[0071] Remove PBS and add DAB colorimetric solution: 5-10 minutes, observe the color, and when the brown color is appropriate, immerse in distilled water to stop the color development, and rinse thoroughly with running water for 5 minutes. DAB solution: Solution A: 40ul + Solution B: 760ul.

[0072] (8) Hematoxylin counterstaining: (for cell nucleus staining)

[0073] Immerse in filtered hematoxylin, dye for 5 minutes, and rinse with running water for 5 minutes (lift up and down 10 times).

[0074] (9) Decolorization with hydrochloric acid and alcohol

[0075] 70% hydrochloric acid alcohol (400ml 70% ethanol + 1ml concentrated hydrochloric acid), lift up and down 12 times, rinse with running water for 5 minutes, and soak in distilled water for 5 minutes.

[0076] 10. Blueing (making the cell nucleus bluer), dehydration, and sealing

[0077] Soak in PBS for 5 min and rinse in running water for 1 min.

[0078] Reverse cylinder movement: 70% ethanol (2 min) → 80% ethanol (2 min) → 90% (2 min) → 95% ethanol (2 min) → → anhydrous ethanol (2 min × 2 times) → xylene II (10 min) → xylene I (10 min).

[0079] 500ul xylene + 500ul neutral resin sealing.

[0080] The experimental results are as follows Figure 5 Shown: Glycyrrhizic acid reduced the proportion of M1 macrophages in prostate tissue of mice with experimental autoimmune prostatitis.

Claims

1. The use of glycyrrhizic acid in the preparation of a drug for preventing or treating prostatitis, wherein the glycyrrhizic acid has a molecular formula of C 42 H 62 O 16 , the chemical structure is: 。 2. The use according to claim 1, characterized in that The application is the application of glycyrrhizic acid in the preparation of medicines for preventing or treating chronic non-bacterial prostatitis.

3. The use according to claim 2, characterized in that The application is to alleviate the inflammatory response and pain symptoms caused by chronic non-bacterial prostatitis.

4. The use according to any one of claims 1 to 3, characterized in that: The application is a drug that can be used clinically and is prepared using glycyrrhizic acid as an active ingredient using a conventional preparation process.

5. The use according to claim 4, characterized in that The drug further comprises a pharmaceutically acceptable carrier, which is selected from diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

6. The drug according to claim 5, characterized in that The medicine is in the form of injection, tablet, powder, granule or capsule.

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