Application of luteoloside in preparation of medicine for preventing or treating renal interstitial fibrosis

Abstract

The invention discloses an application of luteoloside in preparation of a medicine for preventing or treating renal interstitial fibrosis, creatively discovers the new application of the luteoloside in treatment of renal interstitial fibrosis for the first time, and can be applied to development of the medicine for preventing renal interstitial fibrosis. The invention provides a theoretical basis for research and development of anti-renal interstitial fibrosis medicines, provides a brand new scheme for prevention or treatment of renal interstitial fibrosis, and has important transformation significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to the application of luteolin in the preparation of a drug for preventing or treating renal interstitial fibrosis. background

[0003] Luteoloside, also known as Luteolin-7-O-glucoside, is a flavonoid glycoside compound. It was named after the fact that it was first isolated from Reseda odorata L., a plant of the genus Resedaceae. It is mainly found in the leaves of the Lamiaceae plant, the leaves of Nepeta tenuifolia, the leaves of the Leguminosae plant, the leaves of the genus Codonopsis pilosula, the leaves of the Caprifoliaceae plant, the stems and leaves of the Rosaceae plant, and the content of Luteoloside in honeysuckle is relatively high. Luteoloside has a strong respiratory bactericidal effect, lowers cholesterol, and enhances the dilation of capillaries. In addition, Luteoloside also has a certain effect on the central nervous system.

[0004] Chronic kidney disease (CKD) is characterized by the progressive and irreversible loss of renal function and is a serious life-threatening public health problem worldwide. Epidemiological surveys show that CKD is characterized by high morbidity and mortality, with a global incidence as high as 10-15%, and approximately 50,000 to 1 million deaths from CKD each year. In recent years, the incidence of CKD has been increasing year by year due to the prevalence of diabetes, hypertension and obesity, as well as an aging population. Renal interstitial fibrosis (RIF) is the final common outcome of various progressive kidney diseases and a common pathway for the progression of CKD and its transition to end-stage renal disease. Effective prevention and management of renal fibrosis is crucial to the treatment of CKD.

[0005] Although the current existing technologies have made some progress in understanding the pathogenesis of renal fibrosis, effective treatments to prevent the progression of renal fibrosis or reverse the fibrosis that has already formed remain to be explored. Clinical treatments for the progression of CKD are also very limited. Commonly used drugs are angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists, which can reduce proteinuria. However, they also have many side effects, limited scope of use, and some patients still have CKD progression after using many treatments. Therefore, new intervention measures are urgently needed in this field.

[0006] At present, there is no research or report on the correlation between luteolin and anti-renal interstitial fibrosis. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide the use of luteolin in the preparation of drugs for preventing or treating renal interstitial fibrosis, in order to provide a new method for the prevention or treatment of renal interstitial fibrosis in the field. The present invention discovers and verifies for the first time that luteolin can be used to effectively treat renal interstitial fibrosis and has good potential for development into anti-renal interstitial fibrosis drugs.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of luteolin or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form in the preparation of a drug for preventing or treating renal interstitial fibrosis.

[0010] Furthermore, the structural formula of the luteolin is shown in formula (I):

[0011]

[0012] Furthermore, the luteolin inhibits the excessive deposition of extracellular matrix and renal interstitial fibrosis by inhibiting the expression of Fibronectin, Collagen I and / or α-SMA.

[0013] Furthermore, the drug comprises a preventive or therapeutically effective amount of luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0014] Furthermore, the medicine also contains pharmaceutically acceptable adjuvants and / or excipients.

[0015] Furthermore, the dosage form of the drug is an oral dosage form, a parenteral dosage form or a topical dosage form.

[0016] Furthermore, the dosage form of the drug is a solution, a sustained-release agent, a suspension, a granule, a tablet, a capsule, a powder, an emulsion, a syrup or drops.

[0017] 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.

[0018] In a specific embodiment of the present invention, the present invention has proved through scientific experiments that luteolin can reduce the expression levels of mRNA and protein of Fibronectin, Collagen I, and α-SMA, that is, luteolin 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 luteolin can be used to treat the disease of renal interstitial fibrosis and has important application prospects in the treatment of renal interstitial fibrosis.

[0019] In the present invention, the luteoloside is a flavonoid glycoside compound and also an influenza RNA-dependent RNA polymerase inhibitor, and its corresponding molecular formula is C 21 H 20 O 11 , molecular weight 448.38, structural formula as shown in the above formula (I), CAS number 5373-11-5. Luteolin has a strong respiratory bactericidal effect and is the main active ingredient in the Xinjiang unique medicinal material Qinglan for the treatment of tracheitis. In addition, luteolin also has the effect of lowering cholesterol in atherosclerosis and enhancing the dilation of capillaries. At present, there is no research or report on the correlation between luteolin and anti-renal interstitial fibrosis.

[0020] In a second aspect, the present invention provides a pharmaceutical composition.

[0021] Furthermore, the pharmaceutical composition comprises a preventive or therapeutically effective amount of luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0022] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0023] In some embodiments, the pharmaceutically acceptable adjuvants and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used to help the stability of the drug or to help improve the activity of the active ingredient (i.e., the luteolin or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form as described above in the present invention). The substances include but are not limited to: diluents, surfactants, humectants, adhesives, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents. The pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed.

[0024] In some embodiments, the pharmaceutically acceptable adjuvants and / or excipients may additionally contain liquids such as water, saline, glycerol, and ethanol.

[0025] In a third aspect, the present invention provides a pharmaceutical preparation.

[0026] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0027] In some embodiments, the dosage form of the pharmaceutical preparation includes but is not limited to: tablets, pills, powders, granules, capsules, lozenges, syrups, solutions, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, nasal preparations or pulmonary preparations, etc., for patient ingestion.

[0028] In a fourth aspect, the present invention provides an in vitro method for inhibiting the expression of Fibronectin, Collagen I and / or α-SMA for non-therapeutic purposes.

[0029] Furthermore, the method comprises: treating a system in need with luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0030] Furthermore, the system includes a cell system, a subcellular system, a tissue system or an organ system.

[0031] In addition, the present invention also provides a method for preventing or treating renal interstitial fibrosis, the method comprising administering to a subject in need thereof a preventive or therapeutically effective amount of the luteolin as described above or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form, or the pharmaceutical composition as described above, or the pharmaceutical preparation as described above.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] The present invention creatively discovered for the first time the new use of luteolin in the treatment of renal interstitial fibrosis, which can be used in the development of anti-renal interstitial fibrosis drugs. The present invention provides a theoretical basis for the research and development of anti-renal interstitial fibrosis drugs, and provides a new solution for the prevention or treatment of renal interstitial fibrosis, which has important transformational significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Expression of Fibronectin, CollagenⅠ and α-SMAmRNA in each group, where K is the blank group and M is the model group;

[0035] 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;

[0036] 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

[0037] The present invention is further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. Those skilled in the art can understand 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. In order to further explain the present invention, some of the terms involved in the present invention are explained as follows:

[0038] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of luteolin, which means those carboxylates, amino acid addition salts, etc. of the compound (luteolin) described in 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 described in the present invention.

[0039] As used herein, examples of the term "pharmaceutically acceptable salts of luteolin" include, but are not limited to, salts 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.

[0040] In some embodiments, pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metals and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, magnesium, potassium, calcium, etc. Examples of suitable amines are chloroprocaine, choline, N,N'-dibenzylethylenediamine, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

[0041] In some embodiments, base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and isolating the free acid.

[0042] In some embodiments, the salt can be a bisulfate, sulfite, bisulfite, sulfate, nitrate, phosphate, pyrosulfate, pyrophosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, bromide, chloride, iodide prepared from an inorganic acid, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like.

[0043] In some embodiments, representative salts include: hydrobromide, acetate, oxalate, valerate, hydrochloride, sulfate, bisulfate, nitrate, stearate, laurate, oleate, palmitate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, naphthoate, and the like.

[0044] In some embodiments, salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, alkanedioic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, octanoate, isobutyrate, oxalate, propionate, malonate, succinate, suberate, fumarate, maleate, sebacate, mandelate, benzoate, etc.

[0045] In some embodiments, pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations, such as sodium, potassium, calcium, lithium, magnesium, etc., and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as gluconate, arginate, galacturonate, etc.

[0046] As used herein, the term "hydrate" refers to a compound obtained by combining the compound (luteolin) of the present invention with water. Generally, the ratio of the number of water molecules contained in the hydrate of the compound to the number of molecules of the compound in the hydrate is determined. Therefore, the hydrate of the compound can be represented by the general formula R·xH2O, for example, wherein R is the compound (luteolin) and x is a number greater than 0.

[0047] As used herein, the term "solvate" refers to a solvent addition form of a compound containing a stoichiometric or non-stoichiometric solvent, including any solvated form of the compound (lunethol) described in the present invention. Conventional solvents include, but are not limited to, water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc. The compounds described herein can be prepared, for example, in a crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0048] As used herein, the term "prevention or treatment" refers to the medical management of a patient for the purpose of preventing, curing, ameliorating or stabilizing a disease, pathological condition or condition. The term includes active therapy, i.e., treatment specifically for the purpose of ameliorating a disease, pathological condition or condition, and also includes etiological treatment, i.e., treatment for the purpose of removing the cause of the disease, pathological condition or condition in question.

[0049] In some embodiments, the term "prevention or treatment" also includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological state, or condition; the term also includes preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of a related disease, pathological state, or condition; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving a related disease, pathological state, or condition. In a specific embodiment of the present invention, the disease is particularly renal interstitial fibrosis.

[0050] As used herein, the term "effective amount" refers to an amount having a preventive or therapeutic effect or the amount required to produce a preventive or therapeutic effect in a subject. For example, a pharmaceutically effective amount for prevention or treatment refers to the amount of a drug required to produce the desired preventive effect or therapeutic effect, which can be reflected by the results of clinical trial results, model animal studies, and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age, and medication history), the type of disease, and the severity of the disease.

[0051] As used herein, the term "prophylactically or therapeutically effective amount" includes both a "prophylactically effective amount" and a "therapeutically effective amount."

[0052] Wherein, the term "prophylactically effective amount" refers to an amount sufficient to prevent a disease, disorder or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder or condition, or an amount to prevent the recurrence of a disease, disorder or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent, when used alone or in combination with other drugs, that provides a preventive benefit in the process of preventing a disease, disorder or condition. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the preventive effect of other preventive drugs. In a specific embodiment of the present invention, the disease, disorder or condition refers to renal interstitial fibrosis, renal interstitial fibrosis-related disorders or conditions.

[0053] Wherein, the term "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the process of treating a disease, disorder or condition, or an amount that delays or minimizes one or more symptoms associated with a disease, disorder or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, when used alone or in combination with other therapies, that provides a therapeutic benefit in the process of treating a disease, disorder or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents. In a specific embodiment of the present invention, the disease, disorder or condition refers to renal interstitial fibrosis, renal interstitial fibrosis-related disorders or conditions.

[0054] As used herein, the term "subject" includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a human. The terms "human," "patient," and "subject" are used interchangeably herein.

[0055] 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.

[0056] Example Application of luteolin in the treatment of renal interstitial fibrosis

[0057] 1. Fluorescence quantitative qPCR detection of the effect of luteolin on the expression of Fibronectin, Collagen I and α-SMA mRNA related to renal interstitial fibrosis

[0058] 1. Experimental Materials

[0059] 1.1 Main experimental equipment

[0060] 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).

[0061] 1.2 Main experimental reagents

[0062] 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).

[0063] 2. Fluorescence quantitative qPCR experimental method

[0064] 2.1 Total RNA extraction (pipette tips and centrifuge tubes are irradiated and free of RNase)

[0065] 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.

[0066] 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 6 Add 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.

[0067] 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.

[0068] 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).

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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.

[0073] 2.2 Reverse transcription (the gun tip and PCR reaction tube are sterilized by wet heat and are free of RNase)

[0074] 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).

[0075] Table 1 Reaction system

[0076] Components volume 5×RT Buffer 4μL Enzyme Mix 1μL Primer Mix 0.5μL Total RNA 2μg RNase free water Add to 20μL

[0077] 2.2.2 Mix gently and centrifuge

[0078] 2.2.3 Reverse transcription program settings (Veriti PCR instrument) are shown in Table 2.

[0079] Table 2 Reverse transcription program settings

[0080] temperature time 37℃ 15min 98℃ 5min

[0081] 2.3 Quantitative PCR

[0082] 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.

[0083] Table 3 Reaction system

[0084]

[0085] 2.3.2 PCR amplification program settings (fluorescence quantitative PCR instrument) are shown in Table 4.

[0086] Table 4 PCR amplification program settings

[0087]

[0088] 2.3.3 Primers. The primer sequences are shown in Table 5.

[0089] Table 5 Primer sequences

[0090]

[0091] 2.4 Result processing

[0092] The ΔΔCT method was used to process the data, where the relevant calculation formula is as follows:

[0093] For the sample to be tested, ΔCT(A)=CT(target gene, sample to be tested)-CT(internal standard gene, sample to be tested);

[0094] For the control sample, ΔCT(B)=CT(target gene, control sample)-CT(internal standard gene, control sample);

[0095] Then calculate the ΔΔCT value, ΔΔCT = AB;

[0096] According to formula 2 -ΔΔCT to calculate the fold expression.

[0097] 3. Experimental groups and treatment methods for each group

[0098] 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 ); Luteolin group: 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 )+Lactoloside (16μmol·L -1 ).

[0099] 4. Experimental results

[0100] 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 luteolin group were significantly decreased (P<0.05). Figure 1 The above results indicate that luteolin has a therapeutic effect on renal interstitial fibrosis.

[0101] 2. Western Blot detection of the effect of luteolin on the expression of Fibronectin, CollagenⅠ and α-SMA proteins related to renal interstitial fibrosis

[0102] 1. Experimental Materials

[0103] 1.1 Main experimental equipment

[0104] 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).

[0105] 1.2 Main experimental reagents

[0106] 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).

[0107] 1.3 Antibodies

[0108] The main antibodies involved in this example are shown in Table 6.

[0109] Table 6 Antibodies

[0110]

[0111] 2. Western Blot Experimental Method

[0112] 2.1 Total cell protein extraction

[0113] 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.

[0114] 2.2 Protein concentration determination

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 2.3 Protein denaturation

[0121] 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.

[0122] 2.4SDS-PAGE electrophoresis

[0123] 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.

[0124] 2.5 Transfer

[0125] 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.

[0126] 2.6 Immune response

[0127] 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.

[0128] 2.7 Chemiluminescence

[0129] 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.

[0130] 2.8 Western Blot Results and Analysis

[0131] (1) Use Image Pro Plus analysis software to process the saved original images.

[0132] (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.

[0133] 3. Experimental groups and treatment methods for each group

[0134] 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 ); Luteolin group: 10% fetal bovine serum + DMEM high glucose medium + TGF-β1 (10 ng mL -1 )+Lactoloside (16μmol·L -1 ).

[0135] 4. Experimental results

[0136] 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 luteolin group was significantly decreased (P<0.05).

[0137] 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 luteolin group was significantly decreased (P<0.001).

[0138] 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 luteolin group was significantly decreased (P<0.05).

[0139] 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 luteolin. Figure 2-3 The above results indicate that luteolin has a therapeutic effect on renal interstitial fibrosis.

Claims

1. Use of luteolin or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form in the preparation of a drug for preventing or treating renal interstitial fibrosis.

2. The use according to claim 1, characterized in that: The structural formula of the luteolin is shown in formula (I):

3. The use according to claim 1, characterized in that: The luteolin inhibits the excessive deposition of extracellular matrix and renal interstitial fibrosis by inhibiting the expression of Fibronectin, Collagen I and / or α-SMA.

4. The use according to claim 1, characterized in that: The medicine comprises a preventive or therapeutically effective amount of luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

5. The use according to claim 1, characterized in that: The medicament further comprises pharmaceutically acceptable adjuvants and / or excipients.

6. The use according to claim 1, characterized in that: The dosage form of the drug is an oral dosage form, a parenteral dosage form or a topical dosage form.

7. The use according to claim 1, characterized in that: The dosage form of the drug is solution, sustained-release agent, suspension, granule, tablet, capsule, powder, emulsion, syrup or drops.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a preventive or therapeutically effective amount of luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

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

10. A method for inhibiting the expression of Fibronectin, Collagen I and / or α-SMA in vitro for non-therapeutic purposes, characterized in that: The method comprises: treating a system in need with luteolin or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

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