New application of isoquercitrin in preparation of medicine for treating renal interstitial fibrosis

Abstract

The invention discloses a new application of isoquercitrin in preparation of a medicine for treating renal interstitial fibrosis, and finds and verifies that the isoquercitrin can be used for effectively treating the renal interstitial fibrosis through experiments for the first time, and the isoquercitrin is a natural flavonoid compound. The isoquercitrin has the advantages of low toxic and side effects, high safety, high effectiveness and the like, in addition, the isoquercitrin is rich in raw material resource and has good social and economic benefits, a brand new thought and strategy are provided for research and development of renal interstitial fibrosis treatment medicines, and the application prospect in the technical field of renal interstitial fibrosis treatment is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a new use of isoquercetin in preparing a medicine for treating renal interstitial fibrosis. Background Art

[0002] Renal interstitial fibrosis (RIF) is the common pathway and main pathological basis for various kidney diseases to develop into end-stage renal failure. Its main pathological features are excessive accumulation of extracellular matrix (ECM) in the renal interstitium, proliferation of fibroblasts, infiltration of inflammatory cells in the renal interstitium, and atrophy of renal tubules caused by various reasons. The occurrence and development process of renal interstitial fibrosis is roughly as follows: various kidney damage factors lead to activation of tubulointerstitial cells, transformation of tubular epithelial cells, and infiltration of inflammatory cells. A variety of vasoactive substances and cytokines that promote fibrosis are released by activated tubulointerstitial cells and inflammatory cells, affecting the dynamic balance of ECM synthesis and degradation, leading to the accumulation of extracellular matrix components and promoting the occurrence and development of fibrosis.

[0003] With the increasing incidence of chronic kidney disease, renal interstitial fibrosis has become a global public health problem. At present, the clinical treatment of renal interstitial fibrosis is limited, mainly by controlling risk factors such as blood pressure and blood sugar, and using some drugs with renal protective effects to delay the progression of the disease, but it is impossible to fundamentally prevent or reverse the process of renal interstitial fibrosis. Therefore, it is of great significance to develop new drugs for renal interstitial fibrosis. Isoquercetin is a natural flavonoid compound, which is widely found in many medicinal plants such as Amomum villosum, mulberry leaves, Apocynum venetum, Rhizoma truncatum, Moringa leaves, Bidens pilosa, Fire charcoal mother, Cyclocarya paliurus leaves, and Thalictrum stenophyllum, and can also be synthesized artificially. As an active ingredient of medicinal plants, isoquercetin has many pharmacological effects. For example, as an important active ingredient of the traditional Chinese medicine Apocynum venetum, it has the effects of lowering blood pressure, antidepressant, sedative, etc. At present, there is no relevant research or report on the association between isoquercetin and renal interstitial fibrosis. Summary of the invention

[0004] In order to overcome the above technical problems existing in the art, the purpose of the present invention is to provide a new use of isoquercetin in the preparation of a medicament for treating renal interstitial fibrosis. The present invention first discovered that isoquercetin can be used in the effective treatment of renal interstitial fibrosis.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides the use of isoquercetin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing renal interstitial fibrosis.

[0007] Furthermore, the medicine comprises a therapeutically and / or preventively effective amount of isoquercetin or a pharmaceutically acceptable salt thereof.

[0008] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0009] Furthermore, the pharmaceutically acceptable excipients are diluents, binders, surfactants, humectants, adsorption carriers, lubricants, fillers and / or disintegrants.

[0010] Furthermore, the drug is in the form of tablets, capsules, injections, granules, powders, solutions, ointments, suppositories, inhalants, gels, microspheres or aerosols.

[0011] Furthermore, the isoquercetin can significantly reduce the expression of Fibronectin, Collagen I and / or α-SMA associated with renal interstitial fibrosis.

[0012] In the present invention, the isoquercitrin is a natural flavonoid compound, and its corresponding molecular formula is C 21 H 20 O 12 , molecular weight is 464.376, structural formula is shown in formula (I), CAS number is 21637-25-2. In this article, the isoquercetin is the same as isoquercitrin. As an important natural active ingredient, isoquercetin is widely used in medical research to study its mechanism of action in anti-inflammatory, cardiovascular protection and other aspects. Whether isoquercetin has the effect of resisting renal interstitial fibrosis has not been reported.

[0013]

[0014] In some embodiments, the Fibronectin, Collagen Ⅰ (type Ⅰ collagen) and α-SMA (α-smooth muscle actin) are markers of renal interstitial fibrosis. Among them, Fibronectin is an important extracellular matrix protein. In the early stage of renal interstitial fibrosis, its upregulated expression can promote processes such as cell adhesion, migration and proliferation, laying the foundation for subsequent fibrosis processes such as collagen deposition. Collagen Ⅰ is the main component of the extracellular matrix, and its large deposition is an important link in renal interstitial fibrosis leading to renal structural destruction and loss of function. α-SMA is mainly expressed in myofibroblasts, which are the main cell type that produces extracellular matrix. Increased expression of α-SMA means the activation and proliferation of myofibroblasts, thereby promoting the progression of renal interstitial fibrosis.

[0015] In a specific embodiment of the present invention, the present invention has proved through scientific experiments that isoquercetin can reduce the expression levels of mRNA and protein of Fibronectin, Collagen I, and α-SMA, that is, isoquercetin can inhibit the excessive deposition of extracellular matrix, inhibit renal interstitial fibrosis, reduce the activity of myofibroblasts, and slow down the development rate of renal interstitial fibrosis, indicating that isoquercetin can be used to treat the disease of renal interstitial fibrosis and has important application prospects in the treatment of renal interstitial fibrosis.

[0016] In some embodiments, in addition to isoquercetin or a pharmaceutically acceptable salt thereof, the related uses of isoquercetin or its hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of drugs for treating and / or preventing renal interstitial fibrosis also fall within the protection scope of the present invention.

[0017] In the present invention, the pharmaceutically acceptable salt refers to a pharmaceutically acceptable salt of isoquercetin, which means those carboxylates, amino acid addition salts, etc. of the compound (isoquercetin) of the present invention, which are suitable for contact with patients within the scope of reliable medical judgment, do not produce undue toxicity, irritation, allergic reactions, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (if possible) the zwitterionic form of the compound (isoquercetin) of the present invention.

[0018] In some embodiments, examples of pharmaceutically acceptable salts of isoquercetin include, but are not limited to, those having (as counter ions) alkali metal ions such as Li + 、Na + or K + or salts with alkaline earth metal ions such as Mg 2+ or Ca 2+ or any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+ or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0019] A second aspect of the present invention provides a pharmaceutical composition.

[0020] Furthermore, the pharmaceutical composition comprises a therapeutically and / or preventively effective amount of isoquercetin or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the main active ingredient of the pharmaceutical composition is isoquercetin or a pharmaceutically acceptable salt thereof. In addition to isoquercetin, the pharmaceutical composition may also contain other drugs that can be used to treat and / or prevent, or for auxiliary treatment and / or prevention of renal interstitial fibrosis.

[0022] In some embodiments, the other drugs that can be used for the treatment and / or prevention, or for the adjuvant treatment and / or prevention of renal interstitial fibrosis include but are not limited to: angiotensin converting enzyme inhibitors (ACEI) (e.g., captopril, enalapril, etc.), angiotensin II receptor antagonists (ARB) (e.g., losartan, valsartan, etc.), aldosterone antagonists (e.g., spironolactone, eplerenone, etc.), calcium channel blockers (CCB) (e.g., nifedipine, amlodipine, etc.), statins (e.g., atorvastatin, simvastatin, etc.).

[0023] In some embodiments, the other drugs that can be used to treat and / or prevent, or for adjuvant treatment and / or prevention of renal interstitial fibrosis are not limited to the specific drugs listed above in the present invention, and any drug that may be used to treat and / or prevent, or may be used for adjuvant treatment and / or prevention of renal interstitial fibrosis falls within the scope of protection of the present invention.

[0024] A third aspect of the present invention provides a pharmaceutical preparation.

[0025] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0026] Furthermore, the dosage form of the pharmaceutical preparation is a dosage form for enteral administration or a dosage form for parenteral administration.

[0027] Furthermore, the dosage form for administration through the gastrointestinal tract is a solution, granules, tablets, capsules, suspensions, powders, sustained-release agents, effervescent agents, emulsions, syrups, drops or chewable tablets;

[0028] The non-intestinal administration dosage form is an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form or a skin administration dosage form.

[0029] In some embodiments, the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prepared into a dosage form suitable for oral administration, parenteral administration or topical administration by conventional methods currently known in the art, such as by conventional mixing, granulation, coating, solvent or lyophilization methods. The dosage form includes but is not limited to: tablets, capsules, granules, oral emulsions, oral solutions, oral suspensions, sustained-release tablets, injections, powders for injection, emulsions for injection, ointments, gels, suppositories, controlled-release tablets, etc.

[0030] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention may further comprise a pharmaceutically acceptable adjuvant, including but not limited to: a stabilizer, a preservative, a solubilizer, a wetting agent, an emulsifier, a buffer, a sweetener, a colorant, a salt for regulating osmotic pressure, a flavoring agent, a masking agent or an antioxidant. In addition, by adding a component capable of delaying the absorption of the active ingredient to the pharmaceutical composition, the absorption of the pharmaceutical dosage form can be prolonged.

[0031] Those skilled in the art will appreciate that, although the pharmaceutical composition or pharmaceutical preparation mentioned above in the present invention may further contain pharmaceutically acceptable adjuvants and / or excipients, when isoquercetin or its pharmaceutically acceptable salt is used as a drug for humans or animals, they may also be administered in their own form, that is, the present invention may be achieved without adding any of the above-mentioned pharmaceutically acceptable adjuvants and / or excipients.

[0032] In some embodiments, suitable administration methods for the pharmaceutical compositions or pharmaceutical formulations described herein include any of the various methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical compositions or pharmaceutical formulations described herein into the subject, including but not limited to oral administration, topical administration, parenteral administration, administration by inhalation spray, rectal administration, nasal administration, buccal administration, or administration by an implanted reservoir.

[0033] In some embodiments, oral administration or injection administration can be selected, wherein injection administration includes the form of rapid injection or continuous infusion. In the case of a pharmaceutical composition or pharmaceutical preparation for injection administration, it can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it can contain formulation agents, such as preservatives, stabilizers, suspending agents and / or dispersing agents.

[0034] In some embodiments, when the pharmaceutical composition or pharmaceutical preparation is actually used, its administration regimen and dosage regimen can be selected according to a variety of factors, including the type, species, age, weight, sex and type of disease to be treated of the subject; the severity of the disease to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof used. A dosing and / or dosage regimen can be used, for example, to prevent a disease, inhibit (completely or partially inhibit) a disease, or stop the development of the disease. In a specific embodiment of the present invention, the disease specifically refers to the disease of renal interstitial fibrosis.

[0035] In addition, the present invention also provides a medicine kit comprising isoquercetin or a pharmaceutically acceptable salt thereof, that is, isoquercetin or a pharmaceutically acceptable salt thereof is formulated in separate unit preparations, and these separate unit preparations are combined to form a medicine kit. In this case, isoquercetin or a pharmaceutically acceptable salt thereof in the separate unit preparations can be easily administered to a subject simultaneously, sequentially or separately in time.

[0036] In addition, the present invention also provides a method for treating and / or preventing renal interstitial fibrosis, the method comprising the following steps: administering a therapeutically and / or preventively effective amount of isoquercetin or a pharmaceutically acceptable salt thereof, the pharmaceutical composition as described above, or the pharmaceutical preparation as described above to a subject in need thereof.

[0037] In some embodiments, the subject (or subject, or patient) includes one or more animals, including, for example, cattle, horses, sheep, primates, fowl and rodent species. The subject can be a mammal, a bird, a fish, a reptile or an amphibian. The mammal includes a human or a non-human mammal. In other embodiments, the object can be a mouse, a rat, a hamster, a ferret, a gerbil, a rabbit, a monkey, a chimpanzee, a horse, a pony, a donkey, a sheep, a pig, a chicken, a goat, a cat or a dog. In a preferred embodiment, the subject is a human. In a more preferred embodiment, the subject is a patient with renal interstitial fibrosis to be treated and / or prevented.

[0038] In some embodiments, the drug, pharmaceutical composition or pharmaceutical preparation may be administered to the subject by oral administration, injection or topical administration. For example, the method may include administering the drug, pharmaceutical composition or pharmaceutical preparation to the subject three times a day, once a day, once every two days, etc. In some embodiments, injection administration may include subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, injection administration may include injecting the drug, pharmaceutical composition or pharmaceutical preparation directly into the focus of the subject or in an area near the focus. In some embodiments, a combination of different administration methods may also be used.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention discovers for the first time and confirms through experiments that isoquercetin can be used for the effective treatment of renal interstitial fibrosis. Isoquercetin is a natural flavonoid compound with the advantages of low toxicity, high safety, high effectiveness, etc. In addition, the raw material resources of isoquercetin are abundant and have good social and economic benefits. The present invention provides a new idea and strategy for the research and development of drugs for the treatment of renal interstitial fibrosis, and has broad application prospects in the technical field of renal interstitial fibrosis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Expression of Fibronectin, CollagenⅠ and α-SMAmRNA in each group, where K is the blank group and M is the model group;

[0042] Figure 2 : Western Blot detection band diagram of Fibronectin, CollagenⅠ and α-SMA protein in each group, where A represents the blank group, B represents the model group, C represents the quercetin group, and D represents the isoquercetin group;

[0043] Figure 3 : Bar graph of Fibronectin, CollagenⅠ and α-SMA protein expression in each group. Compared with the blank group, ** represents P<0.05, **** represents P<0.001; compared with the model group, * represents P<0.05, *** represents P<0.001. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0045] The drugs, reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0046] Example Application of isoquercetin in the treatment of renal interstitial fibrosis

[0047] 1. Detection of the effect of isoquercetin on the expression of Fibronectin, Collagen I and α-SMA mRNA related to renal interstitial fibrosis by fluorescence quantitative qPCR

[0048] 1. Experimental Materials

[0049] 1.1 Main experimental equipment

[0050] Vortex mixer (SCILOGEX, SCI-VS), handheld centrifuge (SCILOGEX, S1010E), metal bath (SCILOGEX, SCI120-S), film sealer (convenient type) (Wuhan Sevier Biotechnology Co., Ltd., FS-E30), microplate centrifuge (LABGIC, CF2800M), PCR reaction plate (Mona Biotechnology Co., Ltd., MQ50201), fluorescence quantitative PCR sealing film (Mona Biotechnology Co., Ltd., MQ65001), PCR reaction tube (flat cover, enzyme-free) (AXYGEN, PCR-02-C), centrifuge tube 1.5mL (enzyme-free) (AXYGEN, MCT-150-C), ultra-micro spectrophotometer (Thermo, NanoDrop2000), desktop high-speed refrigerated microcentrifuge (SCILOGEX, CF1524R), fluorescence quantitative PCR instrument (Xi'an Tianlong Technology Co., Ltd., TL-988), PCR instrument (Thermo, ABI Veriti).

[0051] 1.2 Main experimental reagents

[0052] TRIzol reagent (Thermo, 15596026), Water Nuclease-Free (Wuhan Saiweier Biotechnology Co., Ltd., G4700), ReverTra qPCR RT Kit (TOYOBO, FSQ-101), Green Realtime PCR Master Mix (TOYOBO, QPK-201), chloroform (Sinopharm Chemical Reagent Co., Ltd., 10006818), isopropanol (Sinopharm Chemical Reagent Co., Ltd., 80109218), and anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 10009218).

[0053] 2. Fluorescence quantitative qPCR experimental method

[0054] 2.1 Total RNA extraction (pipette tips and centrifuge tubes are irradiated and free of RNase)

[0055] 2.1.1 Sample pretreatment. Cells: Take out the cultured six-well plate, gently remove the culture medium with a pipette, avoid touching the bottom of the culture bottle, wash the cells twice with PBS, and remove the excess PBS.

[0056] 2.1.2 Trizol lysis: Add 1 mL of Trizol to each well and let stand for 5 min to lyse the cells (5-10×10 6Add 1 mL Trizol to each cell and pipette several times to fully lyse the cells. Transfer the lysed cell solution to a 1.5 mL RNase-free EP tube.

[0057] 2.1.3 Chloroform (trichloromethane) extraction: Add 200 μL of chloroform (Trizol: chloroform = 5:1) to the enzyme-free EP tube, cover the centrifuge tube, and use a vortex to shake for 15 seconds to mix until the liquid turns light pink, and let it stand for 5 minutes.

[0058] 2.1.4 After centrifugation at 4℃ and 12000rpm for 15min, take out the EP tube and find that the solution is divided into three layers: upper, middle and lower. The upper layer is the aqueous phase (colorless and transparent, containing RNA), and the lower layer is the organic layer (pink, containing DNA and protein, etc.). RNA is mainly concentrated in the transparent and colorless supernatant layer. Pipette the upper colorless aqueous phase (about 500μL) into the prepared 1.5mL RNase-free centrifuge tube (do not aspirate the middle layer).

[0059] 2.1.5 Add 500 μL of isopropanol (equal to the amount of upper water layer) to each EP tube, vortex to mix the liquid (do not shake violently), and then let it stand at room temperature for 10 minutes; after centrifugation at 4°C and 12,000 rpm for 15 minutes, aspirate and discard the supernatant, and retain the white RNA precipitate at the bottom.

[0060] 2.1.6 Wash the precipitate with 75% ethanol: remove the liquid in the tube and use filter paper to absorb the residue, add 1 mL of 75% ethanol solution to each EP tube (prepare for immediate use, DEPC water: anhydrous ethanol = 1:3, mix by inverting after preparation, i.e. 750 μL of anhydrous ethanol, 250 μL of sterile enzyme-free water, in a ratio of 3:1), vortex gently to mix the liquid; centrifuge at 12000g for 5 min at 4°C to allow the RNA precipitate to remain at the bottom of the tube, discard the supernatant, open the EP tube and invert it on a paper towel to let it dry naturally at room temperature for 10 min (be careful to keep it moderately moist to avoid difficulty in dissolving the precipitate due to excessive drying, and find the right balance).

[0061] 2.1.7 RNA drying and dissolution: Add 50 μL DEPC water to each tube, cover the centrifuge tube, vortex to mix, and completely dissolve the precipitate; after 5 minutes in a 65℃ metal bath, immediately place it in a -80℃ freezing box and measure the RNA concentration.

[0062] 2.1.8 RNA concentration determination: Use Nanodrop 2000 to detect RNA concentration and purity: After the instrument is blank and zeroed, take 2.5 μL of the RNA solution to be tested on the detection base, put down the sample arm, and use the software on the computer to start absorbance detection.

[0063] 2.2 Reverse transcription (the gun tip and PCR reaction tube are sterilized by wet heat and are free of RNase)

[0064] 2.2.1 Preparation of reverse transcription reaction system (20 μL reaction system is shown in Table 1, reverse transcription kit catalog number: FSQ-101).

[0065] Table 1 Reaction system

[0066] Components volume 5×RT Buffer 4μL Enzyme Mix 1μL Primer Mix 0.5μL Total RNA 2μg RNase free water Add to 20μL

[0067] 2.2.2 Mix gently and centrifuge

[0068] 2.2.3 Reverse transcription program settings (Veriti PCR instrument) are shown in Table 2.

[0069] Table 2 Reverse transcription program settings

[0070] temperature time 37℃ 15min 98℃ 5min

[0071] 2.3 Quantitative PCR

[0072] 2.3.1 Take 0.1 mL of PCR reaction plate and prepare the reaction system as shown in Table 3 below. Make 3 replicate wells for each sample. Use a film sealer to seal the plate and centrifuge it in a microplate centrifuge.

[0073] Table 3 Reaction system

[0074]

[0075] 2.3.2 PCR amplification program settings (fluorescence quantitative PCR instrument) are shown in Table 4.

[0076] Table 4 PCR amplification program settings

[0077]

[0078] 2.3.3 Primers. The primer sequences are shown in Table 5.

[0079] Table 5 Primer sequences

[0080]

[0081] 2.4 Result processing

[0082] The ΔΔCT method was used to process the data, where the relevant calculation formula is as follows:

[0083] For the test sample, ΔCT(A)=CT(target gene, test sample)-CT(internal standard gene, test sample); for the control sample, ΔCT(B)=CT(target gene, control sample)-CT(internal standard gene, control sample);

[0084] Then calculate the ΔΔCT value, ΔΔCT = AB;

[0085] According to formula 2 -ΔΔCT to calculate the fold expression.

[0086] 3. Experimental groups and treatment methods for each group

[0087] TCMK-1 cells were cultured in a 37°C, 5% CO2 cell culture incubator with DMEM high glucose medium containing 10% fetal bovine serum. After the cells adhered to the wall and fused to 80%, they were randomly divided into 3 groups and stimulated with different drugs according to the groups. The blank group was treated with 10% fetal bovine serum + DMEM high glucose medium; the model group was treated with 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 ); isoquercetin group: 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 )+isoquercetin (80 μmol·L -1 ).

[0088] 4. Experimental results

[0089] RT-PCR results showed that compared with the blank group, the mRNA expression levels of Fibronectin, CollagenⅠ and α-SMA in the model group were significantly increased (P<0.05); compared with the model group, the mRNA expression levels of Fibronectin, CollagenⅠ and α-SMA in the isoquercetin group were significantly decreased (P<0.05). Figure 1 The above results indicate that isoquercetin has a therapeutic effect on renal interstitial fibrosis.

[0090] 2. Western Blot detection of the effect of isoquercetin on the expression of Fibronectin, Collagen I and α-SMA proteins related to renal interstitial fibrosis

[0091] 1. Experimental Materials

[0092] 1.1 Main experimental equipment

[0093] ELISA detector (Nanjing Detie, HBS-1096A), high-speed tissue grinder (Servicebio, KZ-Ⅲ-F), desktop high-speed refrigerated centrifuge (SCILOGEX, CF1524R), vertical electrophoresis tank (BioRad, Mini-PROTEAN), electrophoresis transfer instrument (BioRad, Mini Trans-Blot), decolorization shaker (Qilin Bell, TS-1000), electric constant temperature incubator (Shanghai Xinmiao, GNP-9080BS-Ⅲ), antibody incubation box (4-grid whole membrane) (Servicebio, G9055-4), 300μL multichannel pipette (Thermo, 4660050), analysis software (Media Cybernetics, Image Pro Plus 6.0), chemiluminescence imaging system (Wuhan Mona, QuickChemi 5200).

[0094] 1.2 Main experimental reagents

[0095] High-efficiency RIPA lysis buffer (Beijing Solebow, R0010), BCA protein concentration determination kit (Beijing Solebow, PC0020), SDS-PAGE gel preparation kit (Shanghai Yazyme, PG112), PVDF membrane (0.22 μm) (Millipore, ISEQ00010), Rainbow 245plus broad-spectrum protein marker (Beijing Solebow, PR1930), ECL Plus super-sensitive luminescent liquid (Beijing Solebow, PE0010), 5× Tris-glycine electrophoresis buffer (Beijing Solebow, T1070), 10× electrophoresis transfer buffer (transfer buffer) (Beijing Solebow, D1060), 5× protein loading buffer (containing DTT) (Beijing Solebow, P1040), 20× TBST buffer (Beijing Solebow, T1082), 5× protein loading buffer (Beijing Solebow, P1040), skim milk powder (BD, 232100), universal antibody diluent (Shanghai Yazyme, PS119).

[0096] 1.3 Antibodies

[0097] The main antibodies involved in this example are shown in Table 6.

[0098] Table 6 Antibodies

[0099]

[0100] 2. Western Blot Experimental Method

[0101] 2.1 Total cell protein extraction

[0102] For adherent cells: rinse the cells with PBS 2-3 times, pour out the PBS after the last wash, and use a pipette to absorb as much residual liquid as possible; add an appropriate volume of RIPA lysis buffer (add various protease inhibitors within a few minutes before use) to the culture plate for 3-5 minutes, and shake the culture plate / bottle repeatedly during this period to ensure that the reagent and cells are in full contact; scrape the cells with a cell scraper and transfer them to a 1.5mL centrifuge tube; lyse on ice for 30 minutes, and use a pipette to repeatedly blow and beat during this period to ensure that the cells are completely lysed; centrifuge at 12000rpm, 4℃, for 10 minutes, and collect the supernatant, which is the total protein solution.

[0103] 2.2 Protein concentration determination

[0104] Take the undenatured protein solution and measure the protein concentration using the BCA protein concentration determination kit. For specific methods, refer to the kit instructions.

[0105] 2.2.1 Preparation of working solution: According to the number of standards and samples, add 50 volumes of BCA reagent to 1 volume of Cu reagent (50:1) to prepare BCA working solution, and mix thoroughly (there may be turbidity during mixing, but it will disappear after mixing). BCA working solution is stable at room temperature for 24 hours.

[0106] 2.2.2 Dilute the standard: Take 10 μl of BSA standard and dilute it to 100 μl with PBS to a final concentration of 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, and 20 μl of the standard to the protein standard wells of the 96-well plate and add PBS to make up to 20 μl.

[0107] 2.2.3 Dilute the sample appropriately and add 20 μl to the sample wells of the 96-well plate. Since the pipette has a large error when taking small samples, the points before the standard line may not be very accurate, so try to make the sample point fall behind 1 / 2 of the standard line.

[0108] 2.2.4 Add 200 μl of BCA working solution to each well and place at 37°C for 15-30 minutes. Measure A562 nm with an ELISA reader and calculate the protein concentration based on the standard curve. When using an incubator for incubation, care should be taken to prevent the test results from being affected by water evaporation.

[0109] 2.3 Protein denaturation

[0110] The protein solution was added with 5* reduced protein loading buffer at a ratio of 4:1, denatured in a boiling water bath for 15 min, and stored in a -20°C refrigerator for later use.

[0111] 2.4SDS-PAGE electrophoresis

[0112] Clean the glass plate. Glue preparation and sample loading. After the glass plate is dried naturally, a concave glass plate and a flat glass are paired together, placed in the glue dispenser, and the inclined insert is inserted to fix the glass plate. Check whether the bottom is aligned to avoid glue leakage. According to the experimental requirements, the separation gel of the corresponding concentration is prepared, and after adding TEMED, it is immediately mixed evenly. Pour the separation gel into an appropriate container. You can use a comb to test before pouring it to ensure that the comb teeth are about 5-8mm away from the liquid surface of the separation gel. Then, slowly and evenly add pure water to the upper layer of the separation gel until it is full. After about 30 minutes, when the separation gel solidifies, the upper layer of water can be poured out, and the remaining water can be dried with absorbent paper. Prepare 5% concentrated gel according to the ratio, stir it fully immediately after adding TEMED, and then fill the remaining space with glue, and then insert the comb into the concentrated gel to ensure that no bubbles are generated under the comb. After the concentrated gel solidifies, remove the glue dispenser, carefully pull out the comb, and you can prepare to start electrophoresis. Place the gel caster in the electrophoresis tank, add enough electrophoresis solution, and then load the sample for electrophoresis. Keep the voltage at 200V for about 30 minutes. Stop the electrophoresis when the bottom of the bromophenol blue is about 1cm, and then transfer to the membrane.

[0113] 2.5 Transfer

[0114] Prepare 6 pieces of 7×9cm filter paper and a PVDF (0.45μm) membrane of appropriate size. The PVDF membrane should be activated with methanol for 2 minutes before use. Put the transfer clamp, two sponges, a glass rod, filter paper and activated PVDF membrane in a basin with transfer liquid. Open the clamp, the left side is white and the right side is black, add a sponge and three layers of filter paper on each side. Carefully peel off the separation gel and place it on the filter paper, and stick the PVDF membrane on the gel. There should be no bubbles between the membrane and the gel. Transfer conditions: 300mA constant current, 30min, place the transfer equipment in ice water to cool down during the transfer process.

[0115] 2.6 Immune response

[0116] Place the transferred membrane in an incubation tank filled with TBST, rinse it quickly once, then add protein-free rapid blocking solution milk, place it on a decolorizing shaker, and block it at room temperature for 10 minutes. According to the antibody instructions, dilute the primary antibody. After preparation, pour out the blocking solution in the incubation tank, add the prepared primary antibody, and incubate it on a shaker at 4°C overnight (shake slowly). Recover the primary antibody, rinse the membrane quickly three times with TBST, then add TBST, and place it on a decolorizing shaker for rapid elution, 5 minutes each time, and wash three times. Dilute the secondary antibody with TBST at a ratio of 1:5000, then add it to the incubation tank, place it on a shaker and shake slowly, and incubate it at room temperature for 30 minutes. Rinse the membrane quickly three times with TBST, then add TBST, and place it on a decolorizing shaker for rapid elution, 5 minutes each time, and wash three times.

[0117] 2.7 Chemiluminescence

[0118] Mix ECL A and B solutions in a 1:1 ratio and reserve for later use. Place the PVDF membrane removed from the elution process on absorbent paper, gently absorb the excess liquid on the membrane, and then place the membrane on the shelf of the chemiluminescence instrument. Add the pre-mixed ECL luminescent liquid to ensure that the liquid fully covers the membrane surface. After reacting for 1 minute, gently absorb the excess liquid with absorbent paper. Place the treated membrane into the chemiluminescence instrument, start the chemiluminescence operation according to the preset program, and save the original image after the exposure is completed.

[0119] 2.8 Western Blot Results and Analysis

[0120] (1) Use Image Pro Plus analysis software to process the saved original images.

[0121] (2) Index gray value / internal reference gray value (ratio): The ratio of the index gray value to the internal reference gray value, that is, the relative expression of the sample.

[0122] 3. Experimental groups and treatment methods for each group

[0123] TCMK-1 cells were cultured in a 37°C, 5% CO2 cell culture incubator with DMEM high glucose medium containing 10% fetal bovine serum. After the cells adhered to the wall and fused to 80%, they were randomly divided into 3 groups and stimulated with different drugs according to the groups. The blank group was treated with 10% fetal bovine serum + DMEM high glucose medium; the model group was treated with 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 ); isoquercetin group: 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 )+isoquercetin (80 μmol·L -1 ).

[0124] 4. Experimental results

[0125] Compared with the blank group, the expression of Fibronectin protein in the model group was significantly increased (P<0.01); compared with the model group, the expression of Fibronectin protein in the isoquercetin group was significantly decreased (P<0.05).

[0126] Compared with the blank group, the expression of Collagen Ⅰ protein in the model group was significantly increased (P<0.0001); compared with the model group, the expression of Collagen Ⅰ protein in the isoquercetin group was significantly decreased (P<0.001).

[0127] Compared with the blank group, the expression of α-SMA protein in the model group was significantly increased (P<0.01); compared with the model group, the expression of α-SMA protein in the isoquercetin group was significantly decreased (P<0.05).

[0128] The results showed that TCMK-1 cells underwent significant cell transdifferentiation after being induced by TGF-β1, and the cell transdifferentiation state could be improved after intervention with isoquercetin. Figure 2-3 The above results indicate that isoquercetin has a therapeutic effect on renal interstitial fibrosis.

Claims

1. Use of isoquercetin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing renal interstitial fibrosis.

2. The use according to claim 1, characterized in that: The medicament comprises a therapeutically and / or preventively effective amount of isoquercetin or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients are diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers and / or disintegrants.

5. The use according to claim 1, characterized in that: The dosage form of the drug is tablet, capsule, injection, granule, powder, solution, ointment, suppository, inhalant, gel, microsphere or aerosol.

6. The use according to claim 1, characterized in that: The isoquercetin can significantly reduce the expression of Fibronectin, Collagen I and / or α-SMA related to renal interstitial fibrosis.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically and / or preventively effective amount of isoquercetin or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 7.

9. The pharmaceutical preparation according to claim 8, characterized in that The dosage form of the pharmaceutical preparation is a dosage form for enteral administration or a dosage form for parenteral administration.

10. The pharmaceutical preparation according to claim 9, characterized in that The dosage form for administration through the gastrointestinal tract is a solution, granule, tablet, capsule, suspension, powder, sustained-release agent, effervescent agent, emulsion, syrup, drop or chewable tablet; The non-intestinal administration dosage form is an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form or a skin administration dosage form.

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