Application of isorhamnetin-3-O-neohesperidin in preparation of medicine for preventing and / or treating benign prostatic hyperplasia

Abstract

The invention discloses an application of isorhamnetin-3-O-neohesperidin in preparation of a medicine for preventing and / or treating benign prostatic hyperplasia. In-vitro experiments show that the isorhamnetin-3-O-neohesperidin can inhibit proliferation of prostatic hyperplasia cells, promote apoptosis of the prostatic hyperplasia cells and regulate inflammatory response of the prostatic hyperplasia cells, and rat experiments show that the isorhamnetin-3-O-neohesperidin can improve the hormone endocrine disorder condition of an animal model body, and can inhibit the prostatic hyperplasia cells and promote the prostatic hyperplasia cell apoptosis and regulate the inflammatory response of the prostatic hyperplasia cells. The anti-inflammatory ability of an animal model is enhanced, and the traditional Chinese medicine composition has a relatively good treatment effect on prostatic hyperplasia caused by testosterone propionate. The medicine for preventing and / or treating benign prostatic hyperplasia, provided by the invention, contains a pharmacodynamic dose of isorhamnetin-3-O-neohesperidin and a pharmaceutically acceptable carrier or auxiliary material, can be prepared into a medicine dosage form which is convenient for clinical use and can play an optimal curative effect, and has a relatively high clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and specifically relates to the application of isorhamnetin-3-O-neohesperidin in the preparation of a drug for preventing and / or treating benign prostatic hyperplasia. Background Art

[0002] It is estimated that approximately 50% of men over the age of 50 suffer from benign prostatic hyperplasia (BPH), and its incidence increases with age. BPH is an enlargement of the prostate caused by the accelerated proliferation of stromal and epithelial cells. The excessive cell proliferation mainly affects the transition zone of the prostate, adjacent to the urethra. Compression of the urethra by the enlarged gland gradually leads to urinary tract diseases such as lower urinary tract symptoms (LUTS) and acute urinary retention (AUR). As a result, BPH patients often experience BPH-related symptoms such as incomplete urination, frequency, intermittency, urgency, weak blood flow, spotting, nocturia, etc., and have a poor quality of life. Currently, relying on medication to treat BPH is the preferred treatment for mild to moderate patients.

[0003] Prostatic hyperplasia is caused by the proliferation of prostatic epithelial tissue and stromal tissue. Its pathogenesis is still unclear, but the hypothesis of hormone and inflammation has been proposed. At present, the research on the treatment of prostatic hyperplasia is based on these theoretical guidance.

[0004] The hormonal theory of the cause of prostate disease mainly focuses on the androgen receptor pathway. Testosterone is reduced to dihydrotestosterone under the action of 5α-reductase. Dihydrotestosterone has a higher binding affinity with the androgen receptor. Excessive binding of dihydrotestosterone to the androgen receptor leads to excessive proliferation of prostate cells, which in turn causes benign prostatic hyperplasia.

[0005] Chronic inflammation is another major cause of prostatic hyperplasia. Proinflammatory stimuli promote the growth of prostatic epithelial cells, and in clinical studies, prostatic hyperplasia tissue contains inflammatory infiltration.

[0006] How to prevent or treat prostate hyperplasia is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0007] The purpose of the present invention is to provide the use of isorhamnetin-3-O-neohesperidin in the preparation of a drug for preventing and / or treating benign prostatic hyperplasia.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention claims protection for at least one use of isorhamnetin-3-O-neohesperidin in the following (1)-(6):

[0010] (1) Preparation of drugs for preventing and / or treating benign prostatic hyperplasia;

[0011] (2) preparing drugs for inhibiting the proliferation of prostate hyperplasia cells;

[0012] (3) preparing drugs for inducing and / or promoting apoptosis of prostate hyperplasia cells;

[0013] (4) preparing drugs for regulating the inflammatory response of prostatic hyperplasia cells;

[0014] (5) preparing drugs for inhibiting the activity of prostate hyperplasia cells;

[0015] (6) Preparation of drugs for inhibiting and / or delaying prostatic hyperplasia.

[0016] The chemical structural formula of the isorhamnetin-3-O-neohesperidin is shown below:

[0017]

[0018] In order to solve the problems existing in the prior art, the present invention establishes an in vivo model of benign prostatic hyperplasia in rats, an in vitro BPH-1 cell proliferation inhibition model and an LPS-induced RAW264.7 macrophage inflammation model by testosterone propionate induction method, and uses histological and molecular biological methods to verify that isorhamnetin-3-O-neohesperidin has a strong inhibitory effect on prostate hyperplasia cells and can significantly inhibit the increase of prostate volume, and can be used to prepare drugs for preventing or treating benign prostatic hyperplasia.

[0019] Further: the drug has isorhamnetin-3-O-neohesperidin as the main active ingredient. Further, the content of isorhamnetin-3-O-neohesperidin in the drug is the effective amount. Further, the content of isorhamnetin-3-O-neohesperidin in the drug is 0.01wt%-99.99wt%.

[0020] Furthermore, the medicine also includes other effective ingredients for the treatment or auxiliary treatment of benign prostatic hyperplasia.

[0021] Furthermore, the drug also includes pharmaceutically acceptable carriers or excipients, including but not limited to at least one of excipients, solubilizers, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, and buffers.

[0022] Furthermore, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to at least one of powder injection, injection solution, tablet, pill, capsule, spray and dispersion.

[0023] Furthermore, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

[0024] Furthermore, the dosage of the drug is a pharmaceutically acceptable dosage.

[0025] Beneficial effects of the present invention:

[0026] The present invention shows through in vitro experiments that isorhamnetin-3-O-neohesperidin can inhibit the proliferation of prostate hyperplasia cells, promote the apoptosis of prostate hyperplasia cells, and regulate the inflammatory response of prostate hyperplasia cells. The rat experiment shows that isorhamnetin-3-O-neohesperidin can improve the hormone endocrine disorder of the animal model body, enhance the anti-inflammatory ability of the animal model, and has a good therapeutic effect on prostate hyperplasia caused by testosterone propionate.

[0027] The drug for preventing and / or treating benign prostatic hyperplasia provided by the present invention comprises a pharmaceutically effective amount of isorhamnetin-3-O-neohesperidin and a pharmaceutically acceptable carrier or excipient, and can be prepared into a drug dosage form that is convenient for clinical use and can exert the best therapeutic effect, and has a high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The inhibitory effect of isorhamnetin-3-O-neohesperidin on the proliferation of BPH-1 cells;

[0029] Figure 2 This is the cytotoxicity experiment of isorhamnetin-3-O-neohesperidin on 293T cells;

[0030] Figure 3 The effect of isorhamnetin-3-O-neohesperidin on the cell cycle and apoptosis of BPH-1 cells;

[0031] Figure 4 is the cytotoxicity of isorhamnetin-3-O-neohesperidin on RAW264.7 cells;

[0032] Figure 5 The effect of isorhamnetin-3-O-neohesperidin on the NO content in the culture medium of RAW264.7 cells induced by LPS;

[0033] Figure 6 The effect of isorhamnetin-3-O-neohesperidin on inflammatory factors in RAW264.7 cells induced by LPS;

[0034] Figure 7 To verify the in vivo efficacy of isorhamnetin-3-O-neohesperidin on BPH model rats;

[0035] Figure 8 The effect of isorhamnetin-3-O-neohesperidin on the protein expression in prostate tissue of BPH model rats;

[0036] The significance analysis symbols in the figures are as follows: * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, but not 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 belong to the scope of protection of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the contents of this specification shall prevail.

[0040] Unless otherwise specified, the reagents and materials used in the following examples can be obtained through commercial channels.

[0041] The chemical structural formula of isorhamnetin-3-O-neohesperidin according to the present invention is as follows:

[0042]

[0043] The present invention shows through in vitro experiments that isorhamnetin-3-O-neohesperidin can inhibit the activity of prostate hyperplasia cells, inhibit the proliferation of prostate hyperplasia cells, promote the apoptosis of prostate hyperplasia cells, and regulate the inflammatory response of prostate hyperplasia cells. The rat experiment shows that isorhamnetin-3-O-neohesperidin can improve the hormone endocrine disorder of the animal model body, enhance the anti-inflammatory ability of the animal model, inhibit and / or delay prostate hyperplasia, and has a good therapeutic effect on prostate hyperplasia caused by testosterone propionate.

[0044] The drug for preventing and / or treating benign prostatic hyperplasia provided by the present invention comprises a pharmaceutically effective amount of isorhamnetin-3-O-neohesperidin and a pharmaceutically acceptable carrier or excipient, and can be prepared into a drug dosage form that is convenient for clinical use and can exert the best therapeutic effect, and has a high clinical application value.

[0045] The present invention provides a drug for preventing and / or treating benign prostatic hyperplasia, wherein the drug comprises a pharmaceutically effective amount of isorhamnetin-3-O-neohesperidin, and the drug may further comprise a pharmaceutically acceptable oil-soluble solvent, a stabilizer and a preservative for preparing the drug into a dispersion.

[0046] In other embodiments of the present invention, the drug may further include other medicinal ingredients for the treatment or adjuvant treatment of benign prostatic hyperplasia.

[0047] In other embodiments of the present invention, the drug may also include pharmaceutically acceptable carriers or excipients, including but not limited to excipients, solubilizers, preservatives, stabilizers, wetting agents, emulsifiers, salts for regulating osmotic pressure, buffers, etc. The dosage form of the drug may also be a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection, tablet, pill, capsule, spray, dispersion, etc. The administration route of the drug may be a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation, etc.

[0048] In other embodiments of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage, and the usage and dosage of the drug needs to be determined according to the condition of the disease and can be followed according to the doctor's advice.

[0049] Example 1 Inhibitory effect of isorhamnetin-3-O-neohesperidin on BPH-1 cell proliferation

[0050] 1. Experimental materials:

[0051] Isorhamnetin-3-O-neohesperidin (commercially available), human prostate hyperplasia cells (kindly provided by the School of Pharmacy, China Pharmaceutical University), DMEM high glucose medium (Nanjing KeyGen Biotechnology Development Co., Ltd., batch number: 2367736), FBS special fetal bovine serum (Gibco, batch number: 42G6185K), dimethyl sulfoxide (DMSO, analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20230616), trypsin digestion solution (Biyuntian Biotechnology Co., Ltd., batch number: 011618190313), PBS (Savier Biotechnology Co., Ltd.).

[0052] 2. Experimental instruments:

[0053] BS-124S ​​1 / 10,000 electronic balance (Sartorius, Beijing Sartorius Instrument System Co., Ltd.); FORMA Series II WATER JACKET carbon dioxide incubator (Thermo Scientific corp.); Eppendorf5810R multifunctional desktop centrifuge (Eppendorf, Germany); XDS-1B inverted microscope (Olympus (China) Co., Ltd.); 1300SERIES A2 clean bench (Thermo Scientific corp.).

[0054] 3. Preparation of reagents and drugs

[0055] (1) Preparation of DMEM medium containing 10% fetal bovine serum (FBS):

[0056] Pipette 5 mL of FBS and add it to 45 mL of DMEM culture medium. Store at 4°C.

[0057] (2) MTT preparation:

[0058] Accurately weigh MTT powder and dissolve it in PBS to prepare a 5 mg / mL MTT solution. Vortex and dissolve the solution in a dark place. Sterilize the solution through a 0.22 μm microporous filter membrane and use it immediately after preparation.

[0059] (3) Preparation of isorhamnetin-3-O-neohesperidin solution:

[0060] A certain mass of isorhamnetin-3-O-neohesperidin was weighed and mixed with 0.5% (g / 100 mL) sodium carboxymethyl cellulose solution at concentrations of 0 (blank group), 50 μM, 100 μM, 200 μM, 400 μM, and 800 μM, and the mixture was prepared before use.

[0061] 4. Cell culture and medium replacement

[0062] BPH-1 human prostate hyperplasia cells were cultured at 37°C, 5% CO2, in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics (100U / mL penicillin and 100μg / mL streptomycin). Cell growth was observed under an inverted microscope. Cells were passaged when they were about 80-90% full.

[0063] When the color of the culture medium changes from red to yellow, pour off the culture medium from the non-culture surface, add 2 mL of PBS and wash twice, add 4 to 5 mL of culture medium along the non-culture surface, loosen the culture bottle inoculated with cells slightly, and continue to culture in a 5% CO2, 37°C constant temperature incubator.

[0064] 5. Cell Passaging

[0065] After the cells are 80-90% fused, prepare for subculturing. Prepare new culture medium and PBS, warm them up, and observe the original cells under a microscope; add 2mL PBS on the non-culture surface, shake it gently and pour it out, and remove the remaining liquid with a pipette; add 1mL 0.25% trypsin, shake it gently, and observe the digestion culture under a microscope for about 1 minute. When most of the cells are rounded under an inverted microscope, use a gun to remove the trypsin, add 1mL of complete culture medium containing serum to terminate digestion, shake it gently and pour it out, and remove the remaining liquid with a gun; add 1mL of complete culture medium containing serum each time, blow and beat 20 times (slowly suck and quickly beat) to blow the cells down, transfer the liquid to a 5mL centrifuge tube, and repeat 3 times ; Centrifuge at 1000r / min for 5min, discard the supernatant, and use a pipette to remove the remaining supernatant; add 3mL of new culture medium (1 pass 3 culture) to the centrifuge tube, and blow the cell pellet into a suspension; take another culture bottle, add 4mL of new culture medium, and then add 1mL of cell suspension; stick a label with the cell name, name of the person who subcultured, subculture date and cell generation information on the bottom of the culture bottle; observe the cell morphology under a microscope. If there is no abnormality, place the cell culture bottle flat and carefully move it into the incubator for culture.

[0066] 6. Determination of cell viability (MTT colorimetric assay)

[0067] Collect the cell suspension according to the cell passaging operation, plant 5000 cells (100 μL) in each well of the 96-well plate, place the well plate in the incubator and culture overnight. Add the prepared drugs of different concentrations to the 96-well plate at 100 μL per well, and place it in the incubator for 48 hours. After 48 hours, aspirate the drug solution in the well, add 200 μL PBS to each well to slightly wash the cells and then discard the PBS. Then add 200 μL of the prepared MTT solution to each well, transfer to the incubator and culture for 4 hours. Then carefully aspirate the liquid in the well and add 150 μL DMSO to each well. Oscillate and mix in the dark for 10 minutes, place the well plate in the microplate reader, measure the absorbance value at 490 nm, and calculate the cell proliferation rate. The formula is as follows:

[0068] Cell proliferation rate (%) = (OD490 of the test group / OD490 of the blank group) × 100%.

[0069] The results are as follows Figure 1 As shown, the present invention carried out MTT experiment with BPH-1 cells as the research object, and the results showed that isorhamnetin-3-O-neohesperidin significantly inhibited the proliferation of BPH-1 cells at 25-50 μM (p<0.05), and at 100-400 μM, it could extremely significantly inhibit the proliferation of BPH-1 cells (p<0.01).

[0070] Example 2 Cytotoxicity experiment of isorhamnetin-3-O-neohesperidin on 293T cells

[0071] Human kidney-derived embryonic cells (293T) were used to evaluate drug cytotoxicity, and the MTT experimental method in Example 1 was used to conduct cytotoxicity experiments.

[0072] The results are as follows Figure 2 As shown, the present invention uses human kidney-derived embryonic cells (293T) as the research object to conduct cytotoxicity experiments, and the results show that isorhamnetin-3-O-neohesperidin has no cytotoxicity when the concentration is less than 400 μM.

[0073] Example 3 Flow cytometry detection of the effect of isorhamnetin-3-O-neohesperidin on BPH-1 cell cycle and apoptosis 1. Mitochondrial membrane (JC-10) potential detection

[0074] Take out the cell culture dish that has been treated with drugs, discard the old culture medium, add pre-cooled PBS solution to the well plate to wash the cells, and discard the washing solution. Add EDTA-free trypsin digestion solution to digest the cells, collect the cells and add them to the centrifuge tube, centrifuge at 3000rpm for 5min, collect the cells, and resuspend the cells with 500μL JC-10 staining buffer. Prepare a negative control group that is not treated with drugs and stained. In the prepared positive control tube, only 1μL 10nm mitochondrial oxidative phosphorylation uncoupler is added and incubated on ice for 30min. After incubation, add an equal amount of living cells. Add 1mL JC-10 staining working solution to the positive tube and drug group, mix gently, and incubate at room temperature for 20min. Then centrifuge to collect the cells that have finished staining, wash the cells with PBS, collect the cells by centrifugation, and then resuspend the cells with staining buffer and detect them on the flow machine as soon as possible. In the FITC and PE channels, use CCCP positive tubes and negative tubes to adjust the flow conditions, and then detect the apoptosis rate of the samples.

[0075] 2. Cell cycle and apoptosis detection

[0076] After collecting the cells by trypsin digestion, resuspend the cells with 1 mL of PBS, add 3 mL of anhydrous ethanol to fix the cells, and incubate at 4°C overnight. Collect the fixed cells by centrifugation, discard the supernatant, resuspend the cells with 1 mL of pre-cooled PBS solution, wash the cells by centrifugation, discard the supernatant, add 535 μL of staining buffer, incubate at 37°C in the dark for 30 min, and detect by flow cytometry.

[0077] 3. Cell apoptosis rate detection

[0078] Take out the cell culture dish that has been treated with drugs, absorb the old culture medium into a 15mL centrifuge tube, add pre-cooled PBS solution to the well plate to wash the cells, absorb the washing solution and place it in the centrifuge tube, add EDTA-free trypsin digestion solution to digest the cells, collect the cells and add them to the centrifuge tube, centrifuge at 3000rpm for 5min, collect the cells, and resuspend the cells with 500μL 1×binding buffer. Prepare positive control tubes separately, add only 10μL PI or 5μL Alexa Fluor647, and a negative control tube. Add 10μL PI and 5μL Alexa Fluor 647 to the drug group, mix gently, and detect on the flow machine as soon as possible. After adjusting the flow conditions using the negative tube and two positive tubes, the samples were tested for apoptosis rate.

[0079] The results are as follows Figure 3 As shown, AC: cell cycle distribution; D: Alexa Fluor 647 / propidium iodide double staining method for cell apoptosis detection; E: mitochondrial membrane potential JC-10 for cell apoptosis detection; F: JC-10 staining green / red fluorescence intensity ratio; G: cell apoptosis rate.

[0080] The present invention uses flow cytometry to detect the cell cycle of BPH-1 cells treated with isorhamnetin-3-O-neohesperidin. After drug treatment, the S phase of BPH-1 cells decreased to a certain extent, especially at 200 and 400 μM, there was a very significant decrease (p<0.01). At the same time, the S+G2M phase of each treatment group showed obvious signs of shortening, but there was no significant difference between the drug treatment groups (p>0.05). It shows that from the perspective of the cell cycle, it is verified and explained that isorhamnetin-3-O-neohesperidin can inhibit the proliferation activity of BPH-1 cells.

[0081] The present invention uses flow cytometry to detect the apoptosis rate of BPH-1 cells treated with isorhamnetin-3-O-neohesperidin. After drug treatment, the apoptosis rate of BPH-1 cells increases with the increase of drug concentration, and there are significant differences among the treatment groups (p<0.01), that is, there is a significant dose-effect relationship. The decrease in mitochondrial membrane potential is a specific marker of early apoptosis, which is caused by Figure 3 E shows that the mitochondrial membrane potential of BPH-1 cells treated with isorhamnetin-3-O-neohesperidin decreased significantly, indicating that BPH-1 cells showed premature apoptosis, which is consistent with Figure 3 The increase in Q1 area in each treatment group in D is consistent with the increase in early regulation cells of BPH-1 cells.

[0082] Example 4 Inhibitory effect of isorhamnetin-3-O-neohesperidin on LPS-induced RAW264.7 cell inflammation model 1. Cytotoxicity assay

[0083] The drug cytotoxicity was evaluated using macrophage RAW264.7 cells, and the cytotoxicity experiment was performed using the MTT experimental method in Example 1.

[0084] The results are as follows Figure 4 As shown, isorhamnetin-3-O-neohesperidin had no cytotoxicity to RAW264.7 cells at concentrations of 50-400 μM.

[0085] 2. Model establishment and drug treatment

[0086] RAW264.7 cells were collected according to the cell passage procedure, and PBS was added to suspend the cells to adjust the concentration to 1×10 6 / mL. Inoculate in 12-well plates, 1mL / well. After the cells adhere to the wall, the blank group is cultured with DMEM high-glucose medium, 1mL / well; the experimental group is cultured with 1mL of 10μg / mL LPS. While modeling, culture medium containing corresponding concentrations of drug solution is added to each experimental group. After 24h of culture, the cell morphology is observed, and 50μL of cell culture fluid is taken for cytotoxicity and NO content detection.

[0087] The results are as follows Figure 5 As shown, in the inflammatory cell model, isorhamnetin-3-O-neohesperidin at 50-400 μg / mL significantly reduced the NO content produced by LPS-induced inflammatory cells in the cell culture medium.

[0088] 2. Protein Immunoblotting

[0089] (1) Extraction of cell proteins

[0090] Take out the cell plate after drug administration, discard the Chinese medicine solution in the plate, absorb 1mL of pre-cooled PBS solution to gently wash the cells, then discard the PBS, and repeat the washing steps 2-3 times. Put the prepared cell lysis solution on ice for later use. Place the cell culture plate under a microscope to observe the cell density. After adding an appropriate amount of cell lysis solution according to the number of cells, fully contact the lysis solution with the cells, and place the plate on ice for lysis for 30 minutes. After 30 minutes, stir the liquid in the plate to make it flow naturally to allow the cells to fully lyse. After complete lysis, transfer the sample to a 100℃ metal bath and heat for 15 minutes to completely denature the protein. After heating, vortex and centrifuge the sample, cool to room temperature, and freeze at -20℃.

[0091] (2) Preparation of SDS-PAGE gel

[0092] The specific gel preparation formula is shown in Table 1. Place a clean gel rack on a flat table, fix and clamp the glass plate. Inject the mixed 15% separation gel solution into the gel tank to the red railing on the background of the gel rack. Seal the upper layer with double distilled water and leave it at room temperature for 60 minutes. After the separation gel coagulates, discard the upper layer of double distilled water and absorb the remaining liquid with filter paper. Then add the mixed 5% concentrated gel to the edge of the glass, quickly remove the hole comb, and leave it at room temperature for 30 minutes to allow the concentrated gel to coagulate. After coagulation, the plate gel can be placed in a self-sealing bag, add a small amount of electrophoresis buffer, and store it at 4°C.

[0093] Table 1 SDS-PAGE gel recipe (two gel recipes)

[0094]

[0095] (3) Electrophoresis

[0096] Place two prepared gels into the electrophoresis tank facing each other, and add an appropriate amount of electrophoresis buffer to the tank so that the entire colloid is immersed in the electrophoresis buffer. Pull out the well comb, and slowly add the pre-stained protein standard marker and protein standard (10μg protein content per well) to the respective protein lanes in turn. After loading the samples, cover the power cover according to the principle of "red to red, black to black". According to the needs of different proteins, adjust the concentrated gel electrophoresis voltage to 60V, the time to 20min, and the separation gel voltage to 100V, the time to 120min.

[0097] (4) Transfer

[0098] Prepare to cut out a PVDF membrane of appropriate size, place it in anhydrous methanol to activate for 5 minutes, and then place it in 1×Transfer for equilibrium. Spread the target gel on the transfer filter paper, cover the activated PVDF membrane on the gel, and fit the PVDF membrane tightly, gently push to remove bubbles. Then cover it with transfer filter paper and sponge, close the transfer clip, and place it in the transfer tank. Add 1×Transfer to the transfer tank until the transfer clip is covered, and close the power cover. Transfer for 75 minutes at 4°C and 300mA.

[0099] (5) Closed

[0100] The transferred PVDF membrane was washed with 1×TBST for 3 times, 10 min each time. Then the membrane was placed in 1×TBST solution containing 10% BSA (g / 100 mL) and blocked on a shaker at room temperature for 2 h. After blocking, the PVDF was washed with 1×TBST for 3 times, 10 min each time.

[0101] (6) Antibody incubation

[0102] Place the PVDF membrane in the prepared interleukin-1β and tumor necrosis factor (TNF-α) solution in the antibody incubation box at 4°C overnight. After the primary antibody incubation is completed, the PVDF membrane is shaken and washed three times with 1×TBST, each time for 10 minutes. Place the PVDF membrane in the prepared secondary antibody solution corresponding to the primary antibody in the antibody incubation box and incubate on a shaker at room temperature for 2 hours. After the secondary antibody incubation is completed, the PVDF is shaken and washed four times with 1×TBST, each time for 15 minutes.

[0103] (7) Development

[0104] Place PVDF in a glass dish, draw the mixed chemiluminescent solution onto the PVDF membrane, and make sure the luminescent solution and the PVDF membrane are in full and uniform contact. Use a Tanon developer to expose and develop. Use Image J software to process the obtained bands.

[0105] 4. Extraction of Cellular RNA

[0106] Take out the cell plate after drug administration, discard the drug solution in the plate, absorb 1mL of pre-cooled PBS solution to gently wash the cells, then discard the PBS, and repeat the washing steps 2-3 times. Add 1mL RNATrizol and blow evenly, and transfer to an enzyme-free EP tube. Transfer to a centrifuge and centrifuge at 4°C, 12000rpm for 5min. Pipette 250μL of supernatant into an enzyme-free EP tube, add an equal amount of isopropanol, mix slowly, transfer to a centrifuge, and centrifuge at 4°C, 12000rpm for 5min. After centrifugation, discard the supernatant and blow and wash the tumbling white solid with 1mL 75% (v / v) ethanol. Centrifuge at 7500rpm for 5min, centrifuge, and discard the supernatant. Lay the EP tube flat on the paper to evaporate the liquid. Then use 10μLDEPC water to dissolve the group and blow and mix evenly. Use a multifunctional microplate reader to normalize the concentration of sample mRNA. 10 μL DEPC water, 4 μL 4×DNA wiper, and 2 μL sample were tapped and mixed, centrifuged, and DNA was removed in a gene amplification instrument. Then 4 μL 5×mix was added for reverse transcription. The reverse transcription reaction temperature was set as shown in Table 2. The sample was diluted five times before subsequent use.

[0107] Table 2 Reverse transcription reaction temperature settings

[0108]

[0109] 5. Real-time fluorescence quantitative PCR

[0110] Dissolve the primers to 100 μM according to the instructions, and pipette 10 μL of the front primer and 10 μL of the back primer into 80 μL of DEPC water (primer sequences are shown in Table 3), diluting the primer concentration 10 times. Prepare 10 μL of 2×mix, 4 μL of DEPC water, 1 μL of primer and 5 μL of sample in each EP tube, tap to mix and centrifuge. Add 20 μL of the above mixture to the PCR plate. After adding everything, seal the PCR film and centrifuge to remove bubbles. Put it on the PCR machine, and the qPCR reaction temperature setting is shown in Table 4.

[0111] Table 3 q-PCR primer pairs

[0112]

[0113] Table 4 qPCR reaction temperature settings

[0114]

[0115]

[0116] The results are as follows Figure 6 As shown, A: protein immunoblotting analysis of cell protein inflammatory factor related indicators of different concentrations of drug administration groups; B, C: quantification of Figure A; D: qPCR detection and analysis of cell TNF-α inflammatory factor gene expression of different concentrations of drug administration groups; E: qPCR detection and analysis of cell IL-6 inflammatory factor gene expression of different concentrations of drug administration groups. The present invention detects the expression of inflammatory factors in LPS-induced RAW264.7 cells by different concentrations of isorhamnetin-3-O-neohesperidin by protein immunoblotting and real-time fluorescence quantitative qPCR. Isorhamnetin-3-O-neohesperidin can reduce the protein and gene expression of inflammatory factors TNF-α and IL-6 in LPS-induced inflammatory cells, and can significantly reduce the expression of these two inflammatory factors at a concentration of 200-400 μM. Isorhamnetin-3-O-neohesperidin can play an anti-inflammatory role by reducing the NO content secreted by cells and the expression of inflammatory factors in inflammatory cells.

[0117] Example 7 Verification of the therapeutic effect of isorhamnetin-3-O-neohesperidin on BPH model rats

[0118] 1. Experimental Animals

[0119] 40 male SD rats (180-200 g, Laboratory Animal Management Department of Shanghai Institute of Family Planning Sciences, license number: SCXK (Shanghai) 2018-0006) were used. They were given humane care in accordance with the 3R principles of experimental animal use.

[0120] 2. Drugs and Reagents

[0121] Testosterone propionate (purity: ≥98%, McLean), soybean oil (reagent grade, McLean, batch number: C13043318), finasteride (McLean), sodium carboxymethyl cellulose (CMC-Na, Sinopharm Chemical Reagent Co., Ltd., batch number: 20130314), prostate-specific antigen (PSA) kit, interleukin-6 kit, TNFα kit.

[0122] 3. Instruments

[0123] TB-25 1 / 100,000 electronic balance (Denver Instrument (Beijing) Co., Ltd.); P-214 1 / 10,000 balance (Denver Instrument (Beijing) Co., Ltd.).

[0124] 4. Animal Grouping

[0125] Forty male clean-grade SD rats were randomly divided into five groups: negative control group, model group, positive drug group (finasteride 1 mg kg -1 ), isorhamnetin-3-O-neohesperidin low-dose group (50 mg kg -1 ), isorhamnetin-3-O-neohesperidin high-dose group (100 mg kg -1 ), 8 in each group. During the feeding process, the room temperature was controlled at 20℃~25℃, the humidity was 40%~70%, the light was 12h / d, and the animals were allowed to eat and drink freely. The experiment started after one week of adaptation.

[0126] 5. Modeling and drug administration methods

[0127] 40 normal male rats were fed adaptively for 1 week, 8 rats were randomly selected as blank control group, and the remaining 32 rats were used to establish the model of benign prostatic hyperplasia. The model group was subcutaneously injected with testosterone propionate injection 5mg / kg·d at the back of the neck, and the blank control group was injected with an equal volume of soybean oil for 28 consecutive days. The benign prostatic hyperplasia rat model was established. After 28 days of modeling, drug administration began. The blank group remained unchanged, and the negative control group, model group, and positive drug group (finasteride 1mg·kg -1 ), isorhamnetin-3-O-neohesperidin low and high groups, continuous administration and modeling for 28 days. After 28 days of modeling, oral administration was performed, the blank group and the model group were given 0.5% CMC-Na, and the other groups were given corresponding drugs, and continuous administration and modeling were performed for 28 days.

[0128] 6. Determination of water-loaded urine volume in BPH model rats

[0129] In the first 1-4 weeks of drug administration, after oral administration of the test drug and subcutaneous injection of testosterone propionate injection, each rat was given 30 mL kg of normal saline by oral administration and immediately placed in a metabolic cage with a disposable plastic cup at the bottom to collect and weigh the metabolic urine at the 6th hour, once a week for 4 consecutive weeks.

[0130] 7. Specimen Collection and Processing

[0131] 24 hours after the last administration, the body weight of rats in each group was weighed and recorded. Rats were anesthetized by intraperitoneal injection of 20% (g / 100 mL) urethane. The abdominal cavity was opened and blood was collected through the abdominal aorta. The blood was allowed to stand at room temperature for 1 hour and centrifuged at low temperature (4°C, 3500 r·min). -1 , 10min) to separate serum and store at -80℃. Remove the prostate tissue from both sides of the prostate, carefully remove the surrounding fascia and fat tissue, rinse in pre-cooled saline, wipe dry with filter paper, weigh the prostate weight with an electronic balance, and calculate the prostate index (PI) (PI = prostate wet weight (g) / body weight (g) × 100). Take the dorsolateral lobe of the prostate and fix it in 4% (g / 100mL) paraformaldehyde solution for tissue morphology detection. Carefully remove the mesentery and fat around the urogenital system with surgical instruments, then separate the epididymis and rinse it with 1×PBS. Cut the epididymis into several sections, then squeeze it with tweezers, squeeze the sperm into saline, and incubate it in a cell culture incubator for 20min to allow the sperm to disperse on its own. Take out the culture dish and add physiological saline to dilute the sperm suspension 100 times. Then use a pipette to absorb the suspension and drop it into the gap between the counting platform and the edge of the coverslip of the hemacytometer to allow the suspension to penetrate into and fill the counting chamber. Count the sperm count under an optical microscope.

[0132] 8. HE staining pathological detection

[0133] The dorsolateral lobe of the rat prostate was fixed in 4% paraformaldehyde solution for 48 hours, rinsed with running water for 3 hours, dehydrated with ethanol gradient, transparentized with xylene, embedded in conventional paraffin, cooled at room temperature overnight, cut into blocks and sliced, and made into 4-micron pathological sections. The sections were stained with hematoxylin-eosin and sealed with Zhongxing numerical sealant, and the morphological changes of prostate tissue were observed under an optical microscope.

[0134] The results are as follows Figure 7 As shown, A: rat body weight; B: prostate weight; C: prostate index; D: prostate volume; E: epididymal weight; F: epididymal index; G: fourth week urine output; H: prostate epithelial thickness; I: prostate morphology, prostate pathological tissue section H&E staining, epididymal sperm microscope observation; J: epididymal sperm count. After treatment with each group of drugs, compared with the blank group, each drug treatment group had no effect on the body weight and major organs of the rats, and it was preliminarily determined that each drug had no toxic side effects on the rats. Compared with the model group, the prostate volume and weight of rats were significantly reduced after administration of isorhamnetin-3-O-neohesperidin, which effectively inhibited prostate hyperplasia.

[0135] Compared with the model group, the isorhamnetin-3-O-neohesperidin-treated group significantly reduced the thickness of the prostate epithelium (p<0.01), reduced the number of papillary glands and reduced the degree of protrusion. Moreover, with the increase of the drug concentration, the prostate columnar processes in the high-concentration drug treatment group were significantly reduced, and the glandular epithelial thickness also decreased accordingly.

[0136] Compared with the blank group, the urine output of rats in the model group was significantly inhibited (p<0.001), and the urine output of rats with benign prostatic hyperplasia was significantly increased after treatment with isorhamnetin-3-O-neohesperidin. Compared with the blank group, the epididymal sperm count of rats in the model group was significantly increased (p<0.001), and treatment with isorhamnetin-3-O-neohesperidin had no significant effect on the epididymal sperm count of rats with benign prostatic hyperplasia, indicating that isorhamnetin-3-O-neohesperidin has a therapeutic effect on benign prostatic hyperplasia without the side effects of reduced ejaculation volume or reduced sperm storage.

[0137] 9. Immunohistochemistry for pathological detection

[0138] Prostate tissue sections of rats were taken for immunohistochemical staining to detect the expression of AR protein, PCNA protein and IL-6 protein in the epithelial and interstitial regions of prostate tissue. Under optical microscopy, the cell nucleus was blue, and the protein positive expression was brown-yellow. Five discontinuous high-power microscope fields (400×) were taken, and the average optical density value (IOD / Area) of positive staining in each field was calculated.

[0139] The results are as follows Figure 8 As shown, A: IL-6 protein expression in rat prostate; B: AR protein expression in rat prostate; C: PCNA protein expression in rat prostate; D: quantification of A; E: quantification of B; F: quantification of C.

[0140] Androgen receptor (AR) plays a core role in the proliferation and function of prostate cells. DHT has a higher affinity for binding to AR than testosterone, and activated AR can promote the expression of PCNA to promote the growth and proliferation of prostate cells. The present invention uses immunohistochemistry to detect the protein expression of AR and PCNA in the prostate of BPH rats. Compared with the blank group, the expression of AR and PCNA proteins in the prostate of the model group rats increased significantly (p<0.01), and the isorhamnetin-3-O-neohesperidin treatment group significantly inhibited this increase to a certain extent.

[0141] In BPH, chronic inflammation is considered to be one of the factors that lead to prostate cell proliferation and prostate volume enlargement. There is a relationship between serum TNF-α and IL-6 levels and BPH with histological inflammation. Compared with the blank group, the expression of IL-6 protein in the model group rats increased significantly, and isorhamnetin-3-O-neohesperidin significantly inhibited this increase, alleviating tissue inflammation in the prostate of BPH rats.

[0142] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of isorhamnetin-3-O-neohesperidin in at least one of the following (1)-(6), (1) preparing a drug for inhibiting the activity of prostate hyperplasia cells; (2) preparing drugs for inhibiting the proliferation of prostate hyperplasia cells; (3) preparing drugs for inducing and / or promoting apoptosis of prostate hyperplasia cells; (4) preparing drugs for regulating the inflammatory response of prostatic hyperplasia cells; (5) Preparation of drugs for preventing and / or treating benign prostatic hyperplasia; (6) Preparation of drugs for inhibiting and / or delaying prostatic hyperplasia.

2. The use according to claim 1, characterized in that: The medicine uses isorhamnetin-3-O-neohesperidin as a main active ingredient.

3. The use according to claim 2, characterized in that: The content of isorhamnetin-3-O-neohesperidin in the medicine is the effective amount.

4. The use according to claim 2 or 3, characterized in that: The content of isorhamnetin-3-O-neohesperidin in the medicine is 0.01wt%-99.99wt%.

5. The use according to claim 2 or 3, characterized in that: The medicine also comprises other effective ingredients and is used for the treatment or auxiliary treatment of benign prostatic hyperplasia.

6. The use according to claim 2 or 3, characterized in that: The drug also includes a pharmaceutically acceptable carrier or excipient, and the pharmaceutically acceptable carrier or excipient is at least one of an excipient, a solubilizer, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, and a buffer.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form, and the dosage form is at least one of powder injection, injection solution, tablet, pill, capsule, spray and dispersion.

8. The use according to any one of claims 1 to 6, characterized in that: The administration route of the drug is a pharmaceutically acceptable route, which is oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

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