Separation extraction and new application of trametes robiniophila clear paste neutral water-soluble polysaccharide

Abstract

The invention discloses separation and extraction and novel application of neutral water-soluble polysaccharide in trametes robiniophila clear paste. According to the invention, a water-soluble neutral polysaccharide is extracted from trametes robiniophila clear paste, and the molecular weight of the extract is 20-22kDa. The trametes robiniophila clear paste extract is used for treating the renal interstitial fibrosis, it is proved that the traditional Chinese medicine trametes robiniophila can inhibit the renal interstitial fibrosis in multiple aspects of cells, animals and the like, the trametes robiniophila is a convertible clinical potential medicine, and more benefits are brought to kidney disease patients.

Description

Technical Field

[0001] The invention relates to the technical field of protein extraction, and in particular to the separation, extraction and new application of neutral water-soluble polysaccharides in Sophora auricula paste. Background Art

[0002] Chronic kidney disease (CKD) refers to chronic kidney structural and functional disorders caused by various reasons, including normal and abnormal pathological damage to the glomerular filtration rate, abnormal blood or urine components, and abnormal imaging examinations, or unexplained decrease in glomerular filtration rate (GFR) for more than 3 months. The course of CKD is an irreversible chronic progressive disease, eventually leading to end-stage renal disease (ESRD). ESRD patients need lifelong renal replacement therapy (including maintenance hemodialysis, peritoneal dialysis or kidney transplantation), which not only seriously affects the quality of life of patients, but also brings heavy mental stress and economic burden to patients and their families, and consumes huge social medical resources. Renal interstitial fibrosis (RIF) is a common pathological process of various chronic kidney diseases progressing to end-stage renal failure. The pathological characteristics are that the normal renal interstitial and tubular structures are replaced by a large amount of accumulated extracellular matrix (ECM), such as type I, III, IV collagen, fibronectin (FN), laminin (LN), etc. Therefore, inhibiting the progression of renal interstitial fibrosis is an important way to delay the development of chronic kidney disease patients into ESRD.

[0003] Huaier is the fruiting body of Trametes robiniophila Murr. of the Polyporaceae family. Huaier extract (Huaier) is a hot water extract of Huaier fungus fermentation, and the main active ingredient of its crude extract is polysaccharide protein. Huaier granules have always had good clinical effects in the adjuvant treatment of various cancers or cancer surgery, but the preparation of Huaier extract polysaccharides and related research on renal interstitial fibrosis have not been reported. Summary of the invention

[0004] Based on the above background, the purpose of the present invention is to provide a neutral water-soluble polysaccharide extracted from Sophora auricula paste, and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for separating and extracting neutral water-soluble polysaccharides, the method comprising the steps of:

[0007] (1) removing protein, pigment and small molecules from the aqueous solution of Sophora auricula extract to obtain total polysaccharides; (2) dissolving the total polysaccharides obtained in (1) in water, purifying and eluting, tracking and detecting by phenol-sulfuric acid method, collecting according to peak shape, concentrating, dialyzing and freeze-drying to obtain polysaccharide components; (3) dissolving the polysaccharide components obtained in (2) in water, purifying by chromatography column, collecting symmetrical peaks, concentrating the collected solution by rotary evaporator, and freeze-drying to obtain purified polysaccharides.

[0008] In the present invention, the term "polysaccharide" refers to a sugar chain linked by glycosidic bonds, a polymeric sugar high molecular carbohydrate composed of monosaccharide units. The structure of polysaccharides is usually composed of a variety of monosaccharide units, such as glyceraldehyde for trisaccharides; erythrose and thulose for tetrasaccharides; arabinose, ribose, xylose, and lyxose for pentose; and glucose, mannose, fructose, and galactose for hexose. Polysaccharides composed of the same monosaccharides are homopolysaccharides, and polysaccharides composed of different monosaccharides are heteropolysaccharides.

[0009] The term "neutral polysaccharide" refers to a polymer composed of two or more different glycosyl monomers that do not contain organic acids. Acidic polysaccharides are more complex, containing not only two or more glycosyl monomers, but also one or more uronic acid monomers. Most of the polysaccharides present in natural products are neutral polysaccharides and acidic polysaccharides, and the only alkaline polysaccharide is chitin / chitosan. The term "water-soluble polysaccharide" refers to a class of polysaccharide substances that can be dissolved in water, usually with a higher molecular weight and complex chemical structure. Water-soluble polysaccharides are widely present in nature, including plants, seaweed, fungi, bacteria and animal tissues.

[0010] In the present invention, the method for removing protein can be Sevag method, centrifugal ultrafiltration method, enzymatic method, polyacrylamide gel electrophoresis method, guanidinium chloride method, cell membrane disruption method, etc. In some embodiments, the method for removing protein is Sevag method, and the "Sevag method" refers to a method for removing protein impurities from a mixed system, wherein a mixture containing protein impurities is repeatedly shaken with a chloroform solution added with a small amount of isoamyl alcohol or octanol to denature and precipitate the protein and remove it.

[0011] Furthermore, the specific steps of removing the protein are: precipitation with four times the volume of the solution in anhydrous ethanol at 4°C for 24 hours, dialysis with a 1000Da dialysis bag until the dialysate is clear, and deproteinization with Sevag reagent.

[0012] In the present invention, the method for removing pigments can be column chromatography, adsorption, oxidation, etc. The method for removing small molecules can be ultrafiltration, dialysis, etc. In some embodiments, adsorption is used to remove pigments, and dialysis is used to remove small molecules.

[0013] Furthermore, the specific steps of removing pigments and small molecules are: dialysis for one week with a 1000Da dialysis bag, depigmentation with an AB-8 macroporous resin, dialysis with a 1kD pore size dialysis bag, and freeze drying to obtain total polysaccharides.

[0014] In the present invention, the "purification" method includes step-by-step precipitation, salting-out, metal complex method, cellulose column chromatography, quaternary ammonium salt precipitation, cellulose anion exchanger column chromatography, gel column chromatography and preparative zone electrophoresis. In some embodiments, the purification method is gel column chromatography. The "elution" can be eluted with a constant concentration of eluent or with a linear eluent of different concentrations. In some embodiments, elution is performed using a linear eluent of different concentrations.

[0015] Furthermore, the specific steps of the purification and elution are: heating, vortexing, centrifugation at 12000rpm, loading the supernatant onto a DEAE Sepharose Fast Flow purification column, adjusting the flow rate to 15ml / min of distilled water for elution, and the eluent is water and 0.2M-1.0M NaCl aqueous solution.

[0016] Furthermore, the eluent is water, 0.2M NaCl aqueous solution, 0.5M NaCl aqueous solution, and 1.0M NaCl aqueous solution.

[0017] In the present invention, the chromatography column can be a gel chromatography column or an ion exchange chromatography column. The gel chromatography column can be an agarose gel column, a polyacrylamide gel column, or a dextran gel column.

[0018] Furthermore, the chromatography column is a Sephadex G-200 dextran gel column.

[0019] The second aspect of the present invention provides a neutral water-soluble polysaccharide obtained by the method according to the first aspect of the present invention.

[0020] Furthermore, the monosaccharide composition of the neutral water-soluble polysaccharide includes arabinose, galactose, glucose and mannose, and the molar ratio thereof is 11.4:5.8:10.5:22.3.

[0021] Furthermore, the molecular formula of the neutral water-soluble polysaccharide is:

[0022]

[0023] Furthermore, the weight average molecular weight of the neutral water-soluble polysaccharide is 2.0×10 4 ~2.2×10 4 Da.

[0024] Furthermore, the weight average molecular weight of the neutral water-soluble polysaccharide is 2.17×104 Da.

[0025] In the present invention, the chemical formula of arabinose is C5H 10 O5, a left-handed monosaccharide originally extracted from a colloid secreted by a tree called Arabica by complex chemical and physical methods. It has eight isomers, such as β-D-arabinose, β-L-arabinose, etc. The molecular formula of galactose is C6H 12 O6, often found in dairy products or beets, often in the form of D-galactoside in the brain and nerve tissue. The molecular formula of glucose is C6H 12 O6, in aqueous solution, glucose exists in open-chain form to a small extent, mainly in the form of α- or β-pyranose, and the two can be converted into each other. From aqueous solution, three known forms can be crystallized: α-glucopyranose, β-glucopyranose and α-glucopyranose monohydrate. The molecular formula of mannose is C6H 12 O6, also known as D-mannose, can be prepared by hydrolysis of polysaccharides rich in D-mannose (ivory palm seeds, yeast mannan, etc.). It can also be synthesized by oxidation of D-mannitol (a byproduct of the kelp iodine industry) with hydrogen peroxide in the presence of ferrous ions. It can also be prepared by diastereoisomerization of D-glucose or by carbon chain extension of D-arabinose.

[0026] The third aspect of the present invention provides an application of the neutral water-soluble polysaccharide according to the second aspect of the present invention, wherein the application includes any one of the following:

[0027] 1) Application in the preparation of drugs for treating or delaying renal interstitial fibrosis.

[0028] 2) Application in the preparation of drugs for the treatment of chronic kidney disease.

[0029] 3) Application in studying the mechanism of renal interstitial fibrosis.

[0030] 4) Application in inhibiting macrophage activity.

[0031] 5) Application in inhibiting morphological changes of renal tubular epithelial cells.

[0032] 6) Application in inhibiting macrophage migration.

[0033] 7) Application in the treatment of renal tubular damage.

[0034] 8) Application in changing the levels of epigenetic regulatory factors of renal fibrosis.

[0035] In the present invention, renal interstitial fibrosis (RIF) often occurs in inflammatory and non-inflammatory kidney diseases, and is relevant with the decline of renal excretion function. The fibroblasts occupying the renal interstitium are not only mainly involved in the formation of RIF through the generation of extracellular matrix, but also through the regulatory process. They react to the multiple cytokines released by different cell types. In certain embodiments, the neutral water-soluble polysaccharide provided by the first aspect of the present invention can be used as an instrument to study the physiology and pathological mechanism of RIF.

[0036] Furthermore, the renal interstitial fibrosis includes renal interstitial fibrosis caused by arterial nephrosclerosis, renal interstitial fibrosis caused by benign nephrosclerosis, renal interstitial fibrosis caused by malignant nephrosclerosis, folic acid-induced mouse renal interstitial fibrosis, adenine-induced mouse renal interstitial fibrosis, unilateral ureteral ligation mouse renal interstitial fibrosis, Tgfb1-induced primary mouse renal tubular epithelial cell damage and transdifferentiation, and primary mouse renal tubular epithelial cell damage causing primary mouse macrophage polarization and transdifferentiation.

[0037] Furthermore, the chronic kidney disease includes chronic kidney disease caused by primary glomerulonephritis, hypertensive arteriolar sclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions, ischemic nephropathy, hereditary kidney disease and the like.

[0038] Furthermore, the tubulointerstitial lesions include chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, and drug-induced nephropathy.

[0039] Furthermore, the hereditary kidney disease includes polycystic kidney disease and hereditary nephritis.

[0040] Furthermore, the morphological changes of renal tubular epithelial cells are caused by Tgfb1 stimulation.

[0041] Further, the macrophage migration is caused by renal tubular epithelial cell damage;

[0042] Furthermore, the renal tubular damage is caused by folic acid, adenine and / or unilateral ureteral ligation.

[0043] Furthermore, the epigenetic regulatory factors of fibrosis include one or more of α-sma, COL-1, COL-3, and E-cadherin.

[0044] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0045] In the present invention, "pharmaceutically acceptable carrier" refers to any drug carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be used without excessive toxicity, including non-toxic solid, semi-solid or liquid fillers, diluents, encapsulation materials or any type of formulation aids. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the therapeutic composition can include fluids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0046] Furthermore, the pharmaceutically acceptable carrier includes one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a drug carrier without toxic side effects.

[0047] Furthermore, the dosage form of the drug includes drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments or creams.

[0048] Advantages and beneficial effects of the present invention: 1. The present invention uses the Chinese herbal medicine Sophora japonica urn anti-renal interstitial fibrosis polysaccharide extract to treat renal interstitial fibrosis, and confirms at multiple levels such as cells and animals that the Chinese herbal medicine Sophora japonica urn can inhibit renal interstitial fibrosis. It is a potential drug that can be transformed into clinical practice, bringing more benefits to patients with kidney disease.

[0049] 2. At the cellular level, the drug can significantly inhibit Tgfb1-induced primary mouse renal tubular epithelial cell injury and transdifferentiation, as well as primary mouse renal tubular epithelial cell injury-induced primary mouse macrophage migration, polarization and transdifferentiation. At the animal level, compared with folic acid, adenine and unilateral ureteral ligation (UUO) model mice, the drug can significantly reduce kidney injury and fibrosis in model mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the symmetrical peak diagram of the polysaccharide extracted from Sophora auricula paste by gel column separation.

[0051] Figure 2 This is the peak diagram of neutral polysaccharide A (WHPA) sample.

[0052] Figure 3 This is the peak diagram of neutral polysaccharide B (WHPB) sample.

[0053] Figure 4This is the infrared result of neutral polysaccharide B (WHPB) sample.

[0054] Figure 5 Ion chromatogram (A) and molar ratio diagram (B) of monosaccharide components in neutral polysaccharide B (WHPB).

[0055] Figure 6 This is a graph showing the cell activity detection of Sophora japonica total polysaccharide (THP) on primary mouse renal tubular epithelial cells and primary mouse macrophages.

[0056] Figure 7 This is a graph showing the cell activity detection of total polysaccharide (THP) / neutral polysaccharide (WHP) / neutral polysaccharide A (WHPA) / neutral polysaccharide B (WHPB) on primary mouse macrophages.

[0057] Figure 8 This figure shows the effect of total polysaccharide (THP) / neutral polysaccharide (WHP) in inhibiting Tgfb1-induced primary renal tubular epithelial cell damage.

[0058] Fig. 9 This is a diagram showing the effect of total polysaccharide (THP) / neutral polysaccharide (WHP) in inhibiting macrophage migration caused by primary renal tubular epithelial cell damage.

[0059] Fig.10 This is a diagram showing that total polysaccharide (THP) / neutral polysaccharide (WHP) inhibits renal tubular damage and interstitial fibrosis pathology (PAS, Masson) in folic acid model mice.

[0060] Fig.11 This is a diagram showing that total polysaccharide (THP) / neutral polysaccharide (WHP) inhibits renal tubular damage and interstitial fibrosis pathology (PAS, Masson) in adenine model mice.

[0061] Fig.12 This figure shows that total polysaccharide (THP) / neutral polysaccharide (WHP) inhibits renal tubular injury and interstitial fibrosis pathology (PAS, Masson) in unilateral ureteral ligation (UUO) model mice.

[0062] Fig.13 This is the immunohistochemical staining of neutral polysaccharide (WHP) inhibiting folic acid-induced mouse renal fibrosis-related regulatory factors.

[0063] Fig.14 This is an immunohistochemical staining of neutral polysaccharide (WHP) inhibiting adenine-induced mouse renal fibrosis-related regulatory factors.

[0064] Fig.15 This is the immunohistochemical staining of neutral polysaccharide (WHP) inhibiting the renal fibrosis-related regulatory factors in mice caused by unilateral ureteral ligation (UUO). DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0066] Example 1 Separation, extraction and identification of total polysaccharides and water-soluble polysaccharides from Sophora japonica fungus

[0067] 1. Experimental materials:

[0068] Table 1. Experimental instruments

[0069] Instrument Name factory model Dextran gel GE Superdex-200 GPC-Autopurify fully automatic gel purification system Borui Sugar Bio SHODEX Differential Refractive Index Detector Japan Shodex RID-10A Centrifuge ThermoFisher D-37520 Electronic balance Sartorius CPA225D Automatic Collector Borui BSZ-100 High performance liquid chromatography SHIMADZU RID-10A FRC-10A Ion Chromatography ThermoFisher ICS5000 Electric constant temperature blast drying oven Lichen Technology 101-1BS Nitrogen blowing instrument Lichen Technology UGC-24M

[0070] Table 2. Experimental reagents

[0071] Reagents factory batch number Part Number level Trifluoroacetic acid ACROS A0356762 139725000 AR 50% sodium hydroxide solution Alfa Aesar Z21E036 33382 GR Sodium acetate ThermoFisher 191126 059326 GR

[0072] Table 3. Standard reagents

[0073]

[0074]

[0075] 2. Experimental Methods

[0076] 1. Extraction: Sophora japonica extract (gift from Qidong Gaitianli Pharmaceutical Co., Ltd.) was dissolved in water, precipitated with four times the volume of anhydrous ethanol at 4°C for 24 hours, dialyzed with a 1KD dialysis bag until the dialysate was clear, deproteinized with Sevag reagent, dialyzed with a 1KD dialysis bag for one week, depigmented with AB-8 macroporous resin, dialyzed with a 1KD dialysis bag, and freeze-dried to obtain total polysaccharides (THP). Total polysaccharides (THP) were dissolved in distilled water, heated, vortexed, centrifuged at 12000rpm, and the supernatant was loaded onto a DEAE Sepharose Fast Flow purification column, and eluted with distilled water at a flow rate of 15ml / min. Three groups of solvents were used for elution: three times the volume of the column, 0.2MNaCl, 0.5MNaCl, and 1.0M NaCl. Phenol-sulfuric acid method was used for tracking detection. Detection was performed at 490nm using an enzyme marker. A scatter plot was drawn. According to the peak shape, the samples were collected, concentrated, dialyzed with a 1000Da dialysis bag, and freeze-dried. The obtained components were named as: WHP (water phase neutral polysaccharide), HP-0.2, HP-0.5, HP-1.0. The purpose is mainly to enrich neutral sugars and acidic sugars to prepare for later experiments.

[0077] 2. Separation: Weigh 100 mg WHP, dissolve in 3 mL distilled water, centrifuge at 12000 rpm for 10 min, load the supernatant, purify with a polysaccharide gel purification system specially developed by Boruitang Biotechnology Co., Ltd., and collect online detection with a differential detector (RI-502SHODEX), and collect symmetrical peaks. Concentrate the collected solution with a rotary evaporator, freeze-dry, and obtain polysaccharides separated and purified by gel column.

[0078] 3. Determination:

[0079] 3.1 Determination of polysaccharide molecular weight and purity by HPGPC. Experimental steps: Reagent preparation: 0.05M NaCl solution: accurately prepared, passed through a 0.45μm filter membrane, and ultrasonicated for 10min; Sample and standard solution preparation: accurately weigh the sample and standard, prepare the sample into a 5mg / ml solution, centrifuge at 12000rpm for 10min, filter the supernatant with a 0.22μm microporous filter membrane, and then transfer the sample to a 1.8ml injection vial; Chromatographic method: Chromatographic column: BRT105-104-102 tandem gel column (8×300mm); Mobile phase: 0.05M NaCl solution; Flow rate: 0.6ml / min, column temperature: 40℃; Injection volume: 20μl; Detector: differential detector RD-10A.

[0080] 3.2 Analysis of functional groups of polysaccharides by FT-IR. Experimental steps: 2 mg of sample and 200 mg of potassium bromide were accurately weighed and pressed into tablets. The blank control was made of potassium bromide powder. They were placed in Fourier transform infrared spectrometer FT-IR650 (Tianjin Gangdong Technology Development Co., Ltd.) for scanning and recording.

[0081] 3.3 Determination of monosaccharide composition using ion chromatography. Based on the electrochemical activity of sugar molecules and their ionization in strong alkaline solutions. Sugar compounds are weak acids with pKa>11. In eluents with high pH values, they will exist partially or completely in the form of anions. Based on the differences in ion exchange caused by the differences in pKa of different sugar compounds and the differences in hydrophobicity between certain sugars and anion exchange resins, efficient anion exchange separation of sugar compounds is achieved, and then the current generated by the oxidation reaction of the hydroxyl groups in the sugar molecule structure on the gold electrode surface is detected. Reagent preparation: 15mM NaOH solution 2.4g 50% NaOH solution, 2L water; 5mM NaOH & 100mM NaOAC solution, 1.2g 50% NaOH solution, 8.2g NaOAC, 1L water; Preparation and calculation method of standard solution: Take 16 monosaccharide standards (as shown in Table 3) and prepare about 10mg / ml standard solution. Take each monosaccharide standard solution and accurately prepare 5mg / L gradient concentration standard as Standard. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharide. Sample preparation: Accurately weigh 10mg sample and place it in an ampoule, add 10ml of 3M TFA, and hydrolyze at 120℃ for 3h. Accurately transfer the acid hydrolysis solution to the tube and blow it dry with nitrogen, add 5ml of water and vortex to mix, absorb 100uL and add 900uL deionized water, centrifuge at 12000rpm for 5min. Take the supernatant for IC analysis. Chromatographic method: Chromatographic column: DionexCarbopacTMPA20 (3*150); mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH&100mM NaOAC; flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30℃; detector: electrochemical detector.

[0082] 3.4 Determination of molecular structure by nuclear magnetic resonance. Experimental materials: heavy water (D2O, 99.9%) and deuterated acetone as internal references; freeze dryer, nuclear magnetic resonance instrument Bruker 600M (Nuclear Magnetic Resonance, NMR). Experimental steps: Weigh 50 mg of polysaccharide sample WHPB, dissolve it in 0.5 ml of heavy water and freeze-dry it. Then dissolve the freeze-dried powder in 0.5 ml of heavy water, continue freeze-drying, and repeat the above process to fully exchange active hydrogen. The sample is then dissolved in 0.5 ml of heavy water and placed at room temperature 25°C on a 600MHz nuclear magnetic resonance instrument to measure the 1H NMR spectrum, 13C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0083] 3. Experimental Results

[0084] 1. According to Figure 1The symmetrical part of the first peak, i.e., 100-110 min, was collected as component A. The polysaccharide WHPA was obtained by gel column separation and purification after concentration by rotary evaporator, dialyzation, and freeze drying. The symmetrical part of the second peak, i.e., 125-135 min, was collected as component B. The polysaccharide WHPB was obtained by gel column separation and purification after concentration by rotary evaporator, dialyzation, and freeze drying, with yields of 7.8% and 12.4%, respectively.

[0085] 2. According to Figure 2 , Figure 3 As shown, the calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) are obtained. According to the standard curve, the calculation formula is obtained to calculate the molecular weight of each sample.

[0086] The equation of the lgMp-RT calibration curve is: y = -0.195x + 12.375, R 2 =0.9913;

[0087] The equation of lgMw-RT calibration curve is: y = -0.2078x + 12.968, R 2 =0.993;

[0088] The equation of the lgMn-RT calibration curve is: y = -0.181x + 11.734, R 2 =0.9972.

[0089] Table 4. WHPA sample parameters

[0090] Sample ID RT(min) lgM lm w lgMn Mp M Mn WHPA 41.535 4.3 4.3 4.2 18866 21728 16450

[0091] Table 5. WHPB sample parameters

[0092] Sample ID RT(min) lgM lm w lgMn Mp M Mn WHPB 41.640 4.3 4.3 4.2 17997 20664 15746

[0093] 3. Such as Figure 4 As shown, the infrared results of the polysaccharide component WHPB sample. The absorption band is at 3600-3200cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 There is an absorption peak at 1650cm -1 The absorption peak at 1411 cm -1 There is an absorption peak at 1241cm -1 and 1027cm -1The absorption peak at is attributed to the angular vibration of OH.

[0094] 4. Such as Figure 5 As shown, the monosaccharide composition of WHPB includes arabinose, galactose, glucose, and mannose, and the molar ratio thereof is 11.4:5.8:10.5:22.3.

[0095] 5. The molecular formula of WHPB determined by NMR is as follows:

[0096]

[0097] Example 2 Comparison of the efficacy of total polysaccharide of Sophora japonica urn and neutral water-soluble polysaccharide in protecting renal tubules

[0098] 1. Experimental materials: Sophora japonica total polysaccharide (THP) and neutral water-soluble polysaccharide (WHP) extracted and separated in Example 1, purified neutral water-soluble polysaccharides WHPA and WHPB, primary mouse renal tubular epithelial cells, primary mouse macrophages, C57 / BL-6 mice, and ICR mice.

[0099] 2. Experimental Methods

[0100] 1. The CCK8 experiment was used to detect the activity of the traditional Chinese medicine Sophora japonica total polysaccharide (THP) at the primary mouse renal tubular cell level.

[0101] The process is as follows: primary mouse renal tubular epithelial cells were cultured using DMEM medium (containing 10% fetal bovine serum + 1% penicillin-streptomycin mixture), cell growth was observed, the supernatant was discarded, trypsin digestion was performed, centrifugation was performed at 800rpm for 5 minutes, complete medium was added, and the cells were mixed by blowing to make a single cell suspension, the number of cells was counted using a cell counting plate, 15,000 cells / well were inoculated in a 96-well plate, and 3 replicates were set for each group. The cells were placed in a constant temperature incubator for overnight culture (37°C, 5% CO2), and after starvation for 24 hours, serum-free culture media with different drug concentrations were prepared (the concentration gradient was set to 0,

[0102] 100, 200, 400, 800, 1600 μg / mL), add culture medium (200 μL / well), set up 3 replicates for each group; after culturing for 24 h, replace with CCK8 solution, incubate for 1-2 h, place on a microplate reader to measure the absorbance at 450 nm, and calculate the cell viability.

[0103] 2. The CCK8 experiment was used at the primary mouse macrophage level to detect the activity of different extracted polysaccharide components from the traditional Chinese medicine Sophora japonica.

[0104] The process is as follows: primary mouse renal tubular epithelial cells were cultured using DMEM medium (containing 10% fetal bovine serum + 15% L929 cell culture supernatant + 1% penicillin-streptomycin mixture), the cell growth was observed, the supernatant was discarded, trypsin digestion was performed, centrifugation was performed at 800 rpm for 5 min, complete culture medium was added and the cells were blown and mixed to make a single-cell suspension, the number of cells was counted using a cell counting plate, 15,000 cells / well were inoculated in a 96-well plate, and 3 replicate wells were set up for each group. The cells were placed in a constant temperature incubator and cultured overnight (37°C, 5% CO2). After starvation for 24 hours, culture media with different drug concentrations were prepared (the concentration gradient was set to 0, 100, 200, 400, 800, and 1600 μg / mL), and culture media (200 μL / well) were added. Three replicate wells were set up for each group. After 24 hours of culture, the solution was replaced with CCK8 solution. After incubation for 1-2 hours, the solution was placed in an enzyme reader to measure the absorbance at 450 nm and calculate the cell viability.

[0105] 3. Primary mouse renal tubular epithelial cells were divided into four groups: PBS control group, PBS+THP / WHP control group, Tgfb1 stimulation group, and THP / WHP+Tgfb1 group, and their morphology was photographed under a light microscope.

[0106] 4. Primary mouse renal tubular epithelial cells were co-cultured with primary mouse macrophages. The primary mouse renal tubular epithelial cells were plated in the lower layer, and the primary mouse macrophages were plated in the upper chamber. Tgfb1 stimulated the primary mouse renal tubular epithelial cells in the lower layer, and the downward migration of the upper layer cells was observed. The cells were divided into four groups: PBS control group, PBS+WHP control group, Tgfb1 stimulation group, and WHP+Tgfb1 group. The migration of primary mouse macrophages was photographed under an inverted microscope.

[0107] 5. At the mouse (C57 / BL-6) level, PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide WHP on inhibiting folic acid-induced tubular damage and interstitial fibrosis.

[0108] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, and the groups were: sodium bicarbonate solution intraperitoneal injection group, folic acid model group, WHP intraperitoneal injection treatment folic acid model group. After 1 week, the mice were anesthetized and killed by cervical dislocation, fixed in 10% formalin, sectioned at 4pm paraffin, and stained with PAS and Masson. PAS staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with periodic acid for 20 min, wash with water; stain with hexamine silver stain at 60°C for 30-50 min, until the basement membrane turns black under a microscope; (hexamine silver stain: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops of 4% boric acid, prepare before staining, place in a 60°C oven, add 1 ml 5% silver nitrate after 20 min); add a few drops of 2% gold chloride on the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid and ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with eosin for 1 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum. Masson staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with potassium dichromate for 20 min, wash with water; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with Masson solution for 20 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum.

[0109] 6. At the mouse (ICR) level, PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting adenine-induced renal tubular damage and interstitial fibrosis.

[0110] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, and the groups were: sodium carboxymethylcellulose gavage group, adenine gavage model group, WHP intraperitoneal injection treatment adenine model group. After 4 weeks, the mice were anesthetized and killed by cervical dislocation, fixed in 10% formalin, sectioned at 4pm in paraffin, and stained with PAS and Masson. PAS staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with periodic acid for 20 min, wash with water; stain with hexamine silver stain at 60°C for 30-50 min, until the basement membrane turns black under a microscope; (hexamine silver stain: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops of 4% boric acid, prepare before staining, place in a 60°C oven, add 1 ml 5% silver nitrate after 20 min); add a few drops of 2% gold chloride on the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid and ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with eosin for 1 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum. Masson staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with potassium dichromate for 20 min, wash with water; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with Masson solution for 20 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum.

[0111] 7. At the mouse (C57 / BL-6) level, PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide in inhibiting UUO-induced tubular damage and interstitial fibrosis.

[0112] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, and the groups were: sham operation group, unilateral ureteral ligation (UUO) model group, and unilateral ureteral ligation (UUO) model group treated with Sophora japonica extract (6g / kg / d) gavage. After 1 week, the mice were anesthetized and killed by cervical dislocation, fixed in 10% formalin, sectioned at 4pm in paraffin, and stained with PAS and Masson. PAS staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with periodic acid for 20 min, wash with water; stain with hexamine silver stain at 60°C for 30-50 min, until the basement membrane turns black under a microscope; (hexamine silver stain: 22 ml distilled water + 6 ml 15% hexamethylenetetramine + 4.5 ml 5% borax + 3 drops of 4% boric acid, prepare before staining, place in a 60°C oven, add 1 ml 5% silver nitrate after 20 min); add a few drops of 2% gold chloride on the tissue, wash with 5% sodium thiosulfate; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid and ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with eosin for 1 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum. Masson staining: dewax twice in xylene, 10 min each time; hydrate twice in anhydrous ethanol, 1 min each time; rinse with tap water for 2-3 min; oxidize with potassium dichromate for 20 min, wash with water; stain the nucleus with hematoxylin for 1 min, wash with water; differentiate with 1% hydrochloric acid ethanol for 2-3 s, wash with water; blue with ammonia for 2-3 s, wash with water; stain with Masson solution for 20 min, wash with water; dehydrate with gradient alcohol, make transparent with xylene, and seal with gum.

[0113] 8. At the mouse (C57 / BL-6) level, immunohistochemical staining was used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting folic acid-induced tubular damage and interstitial fibrosis.

[0114] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, and the groups were divided into: sodium bicarbonate solution intraperitoneal injection group, folic acid model group, WHP intraperitoneal injection treatment folic acid model group. After 1 week, the mice were anesthetized and killed by dislocation of the neck, fixed in 10% formalin, and sliced ​​in paraffin at 4pm for immunohistochemical staining. Immunohistochemical staining: Dewaxing: dewaxing in xylene twice, 10 minutes each time; gradient alcohol dehydration: dehydration in 100%, 95%, 80%, and 70% alcohol cylinders, 1 minute each; rinse in tap water for about 10 minutes; PBS wash: 3 minutes each time × 3 times; add endogenous peroxidase blocker to block endogenous peroxidase, in a wet box at room temperature for 10 minutes; PBS wash: 3 minutes each time × 3 times; microwave antigen repair: change the iron slice rack to a plastic slice rack and place it in a citric acid container. In the glass staining jar of sodium citrate buffer, the sodium citrate buffer is fully submerged in the slices to avoid exposing the tissue outside the citrate buffer. Microwave at medium speed for 4 minutes 4 times; after the microwave repair is completed, take the glass jar out of the microwave and place it at room temperature to cool naturally; wash with PBS: 3 minutes each time × 3 times; serum blocking: take the slices out of the glass jar, wipe the moisture around the tissue in the slices with toilet paper, and keep the tissue moist. Place the slices in a wet box, add goat serum for blocking, and incubate at room temperature for 20-30 minutes. Shake off the blocking serum, add the pre-prepared primary antibody while keeping it moist, and incubate overnight at 4°C in the wet box. Take the wet box out of the refrigerator the next day, place it for 30 minutes, and wash with PBS: 3 minutes each time × 3 times; according to the species of the primary antibody, select the corresponding horseradish peroxidase-labeled secondary antibody, prepare the secondary antibody with PBS at a ratio of 1:100, add the secondary antibody, and incubate at 37°C for 40 minutes at room temperature. Wash with PBS: 3 minutes each time × 3 times; DAB color development: prepare DAB color development solution, mix thoroughly, add DAB color development solution to the slice, observe under a microscope, and terminate the reaction with tap water when the color of the tissue turns brown; hematoxylin counterstaining, 30 seconds, rinse with tap water; gradient alcohol dehydration: dehydrate in 80%, 95%, and 100% alcohol cylinders for 2 minutes each. Xylene clearing: clear in xylene twice, each for 5 minutes; seal the slide with neutral gum, observe and take pictures under a microscope.

[0115] 9. At the mouse (ICR) level, PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide on inhibiting adenine-induced renal tubular damage and interstitial fibrosis.

[0116] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, divided into: sodium carboxymethylcellulose gavage group, adenine gavage model group, HP-W intraperitoneal injection treatment adenine model group, 4 weeks after anesthesia, the mice were killed by dislocation of the neck, fixed in 10% formalin, 4pm paraffin section, immunohistochemical staining. Immunohistochemical staining: Dewaxing: dewaxing in xylene twice, 10 minutes each time; gradient alcohol dehydration: dehydration in 100%, 95%, 80%, 70% alcohol cylinders, 1 minute each; rinse in tap water for about 10 minutes; PBS wash: 3 minutes each time × 3 times; add endogenous peroxidase blocker to block endogenous peroxidase, in a wet box at room temperature for 10 minutes; PBS wash: 3 minutes each time × 3 times; microwave antigen repair: change the iron slice rack to a plastic slice rack and place it in a citric acid bath In the glass staining jar of sodium citrate buffer, the sodium citrate buffer is fully submerged in the slices to avoid exposing the tissue outside the citrate buffer. Microwave at medium speed for 4 minutes 4 times; after the microwave repair is completed, take the glass jar out of the microwave and place it at room temperature to cool naturally; wash with PBS: 3 minutes each time × 3 times; serum blocking: take the slices out of the glass jar, wipe the moisture around the tissue in the slices with toilet paper, and keep the tissue moist. Place the slices in a wet box, add goat serum for blocking, and incubate at room temperature for 20-30 minutes. Shake off the blocking serum, add the pre-prepared primary antibody while keeping it moist, and incubate overnight at 4°C in the wet box. Take the wet box out of the refrigerator the next day, place it for 30 minutes, and wash with PBS: 3 minutes each time × 3 times; according to the species of the primary antibody, select the corresponding horseradish peroxidase-labeled secondary antibody, prepare the secondary antibody with PBS at a ratio of 1:100, add the secondary antibody, and incubate at 37°C for 40 minutes at room temperature. Wash with PBS: 3 minutes each time × 3 times; DAB color development: prepare DAB color development solution, mix thoroughly, add DAB color development solution to the slice, observe under a microscope, and terminate the reaction with tap water when the color of the tissue turns brown; hematoxylin counterstaining, 30 seconds, rinse with tap water; gradient alcohol dehydration: dehydrate in 80%, 95%, and 100% alcohol cylinders for 2 minutes each. Xylene clearing: clear in xylene twice, each for 5 minutes; seal the slide with neutral gum, observe and take pictures under a microscope.

[0117] 10. At the mouse (C57 / BL-6) level, PAS and Masson staining were used to detect the effect of Sophora japonica neutral polysaccharide in inhibiting UUO-induced tubular damage and interstitial fibrosis.

[0118] The process is as follows: 20 mice were randomly divided into 4 groups, 5 mice in each group, and the groups were divided into: sham operation group, unilateral ureteral ligation (UUO) model group, WHP intraperitoneal injection treatment unilateral ureteral ligation (UUO) model group. After 1 week, the mice were anesthetized and killed by dislocation of the neck, fixed in 10% formalin, and sliced ​​in paraffin at 4pm for immunohistochemical staining. Immunohistochemical staining: Dewaxing: dewaxing in xylene twice, 10 minutes each time; gradient alcohol dehydration: dehydration in 100%, 95%, 80%, and 70% alcohol cylinders, 1 minute each; rinse in tap water for about 10 minutes; PBS wash: 3 minutes each time × 3 times; add endogenous peroxidase blocker to block endogenous peroxidase, in a wet box at room temperature for 10 minutes; PBS wash: 3 minutes each time × 3 times; microwave antigen repair: change the iron slice rack to a plastic slice rack and place it in a citric acid bath In the glass staining jar of sodium citrate buffer, the sodium citrate buffer is fully submerged in the slices to avoid exposing the tissue outside the citrate buffer. Microwave at medium speed for 4 minutes 4 times; after the microwave repair is completed, take the glass jar out of the microwave and place it at room temperature to cool naturally; wash with PBS: 3 minutes each time × 3 times; serum blocking: take the slices out of the glass jar, wipe the moisture around the tissue in the slices with toilet paper, and keep the tissue moist. Place the slices in a wet box, add goat serum for blocking, and incubate at room temperature for 20-30 minutes. Shake off the blocking serum, add the pre-prepared primary antibody while keeping it moist, and incubate overnight at 4°C in the wet box. Take the wet box out of the refrigerator the next day, place it for 30 minutes, and wash with PBS: 3 minutes each time × 3 times; according to the species of the primary antibody, select the corresponding horseradish peroxidase-labeled secondary antibody, prepare the secondary antibody with PBS at a ratio of 1:100, add the secondary antibody, and incubate at 37°C for 40 minutes at room temperature. Wash with PBS: 3 minutes each time × 3 times; DAB color development: prepare DAB color development solution, mix thoroughly, add DAB color development solution to the slice, observe under a microscope, and terminate the reaction with tap water when the color of the tissue turns brown; hematoxylin counterstaining, 30 seconds, rinse with tap water; gradient alcohol dehydration: dehydrate in 80%, 95%, and 100% alcohol cylinders for 2 minutes each. Xylene clearing: clear in xylene twice, each for 5 minutes; seal the slide with neutral gum, observe and take pictures under a microscope.

[0119] 3. Experimental Results

[0120] 1. By Figure 6 , Figure 7 It can be seen that the total polysaccharide THP extracted from Huaierqing Gao has no inhibitory effect on the activity of normal renal tubular epithelial cells in mice ( Figure 6 ), while inhibiting macrophage activity ( Figure 7 A in the figure); the extracted neutral polysaccharide WHP has twice the inhibitory efficiency on macrophage activity as THP ( Figure 7 B), and neutral polysaccharide WHPA ( Figure 7C) and WHPB( Figure 7 D) has the same inhibitory effect as WHP, and there is no difference between the two ( Figure 7 Therefore, only one of them was selected as a representative for subsequent experiments.

[0121] 2. By Figure 8 , Fig. 9 It can be seen that THP / WHP can effectively inhibit the morphological changes of primary mouse renal tubular epithelial cells induced by Tgfb1 stimulation ( Figure 8 ) and macrophage migration induced by primary mouse renal tubular epithelial cell injury ( Fig. 9 ). And WHP (800μg / mL) only needs half the dose of THP (1600μg / mL) to achieve the same effect.

[0122] 3. By Figure 10-12 It can be seen that THP / WHP can effectively inhibit folic acid ( Fig.10 ), adenine ( Fig.11 ) and unilateral ureteral ligation ( Fig.12 )-induced renal tubular damage and renal interstitial fibrosis in mice, and WHP (0.085 g / kg / d) only required half the dose of THP (0.17 g / kg / d) to achieve the same effect.

[0123] 4. By Figure 13-15 It can be seen that WHP can effectively inhibit folic acid ( Fig.13 ), adenine ( Fig.14 ) and unilateral ureteral ligation ( Fig.15 ) caused an increase in α-sma, COL-1, and COL-3 and a decrease in E-cadherin in the mouse kidneys.

[0124] The above results show that the neutral water-soluble polysaccharides WHP, WHPA and WHPB extracted from Sophora auricularia auricula paste by the extraction and separation method provided by the present invention can treat renal interstitial fibrosis caused by various reasons, and the therapeutic effect is twice that of the total polysaccharides crudely extracted from Sophora auricularia auricula.

[0125] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for separating and extracting neutral water-soluble polysaccharides, characterized in that: The steps of the method include: (1) removing protein, pigment and small molecules from the aqueous solution of Sophora auricula extract to obtain total polysaccharides; (2) dissolving the total polysaccharide obtained in (1) in water, purifying and eluting, performing tracking detection by the phenol-sulfuric acid method, collecting according to the peak shape, concentrating, dialyzing, and freeze-drying to obtain polysaccharide components; (3) The polysaccharide component obtained in (2) is dissolved in water, purified by chromatography, and the symmetrical peak is collected. The collected solution is concentrated by rotary evaporator and freeze-dried to obtain purified polysaccharide.

2. The method according to claim 1, characterized in that The specific steps of removing protein are: precipitation with four times the volume of the solution in anhydrous ethanol at 4°C for 24 hours, dialysis with a 1000Da dialysis bag until the dialysate is clear, and deproteinization with Sevag reagent; Preferably, the specific steps of dialysis to remove pigments and small molecules are: dialysis for one week with a 1000Da dialysis bag, depigmentation with an AB-8 macroporous resin, dialysis with a 1kD pore size dialysis bag, and freeze drying to obtain total polysaccharides; Preferably, the specific steps of purification and elution are: heating, vortexing, centrifuging at 12000rpm, loading the supernatant onto a DEAE Sepharose Fast Flow purification column, adjusting the flow rate to 15ml / min of distilled water for elution, and the eluent is water, 0.2M-1.0M NaCl aqueous solution; Preferably, the eluent is water, 0.2M NaCl aqueous solution, 0.5M NaCl aqueous solution, 1.0M NaCl aqueous solution; Preferably, the chromatography column is a Sephadex G-200 dextran gel column.

3. A neutral water-soluble polysaccharide obtained by the method according to any one of claims 1-2.

4. The neutral water-soluble polysaccharide according to claim 3, characterized in that The monosaccharide composition of the neutral water-soluble polysaccharide includes arabinose, galactose, glucose, and mannose, and the molar ratio thereof is 11.4:5.8:10.5:22.3; preferably, the molecular formula of the neutral water-soluble polysaccharide is:

5. The neutral water-soluble polysaccharide according to claim 4, characterized in that The weight average molecular weight of the neutral water-soluble polysaccharide is 2.0×10 4 ~2.2×10 4 Da; Preferably, the weight average molecular weight of the neutral water-soluble polysaccharide is 2.17×10 4 Da.

6. The use of the neutral water-soluble polysaccharide according to any one of claims 3 to 5, characterized in that: The application includes any of the following: 1) Application in the preparation of drugs for treating or delaying renal interstitial fibrosis; 2) Application in the preparation of drugs for treating chronic kidney disease; 3) Application in studying the mechanism of renal interstitial fibrosis; 4) Application in inhibiting macrophage activity; 5) Application in inhibiting morphological changes of renal tubular epithelial cells; 6) Application in inhibiting macrophage migration; 7) Application in the treatment of renal tubular injury; 8) Application in changing the levels of epigenetic regulatory factors of renal fibrosis.

7. The use according to claim 6, characterized in that: The renal interstitial fibrosis includes renal interstitial fibrosis caused by arterial nephrosclerosis, renal interstitial fibrosis caused by benign nephrosclerosis, renal interstitial fibrosis caused by malignant nephrosclerosis, folic acid-induced mouse renal interstitial fibrosis, adenine-induced mouse renal interstitial fibrosis, unilateral ureteral ligation mouse renal interstitial fibrosis, Tgfb1-induced primary mouse renal tubular epithelial cell damage and transdifferentiation, and primary mouse renal tubular epithelial cell damage causing primary mouse macrophage polarization and transdifferentiation.

8. The use according to claim 6, characterized in that: The chronic kidney disease includes chronic kidney disease caused by primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions, ischemic nephropathy, hereditary nephropathy, etc.; Preferably, the tubulointerstitial lesions include chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, and drug-induced nephropathy; Preferably, the hereditary kidney disease includes polycystic kidney disease and hereditary nephritis.

9. The use according to claim 6, characterized in that: The morphological changes of renal tubular epithelial cells are caused by Tgfb1 stimulation; Preferably, the macrophage migration is caused by renal tubular epithelial cell damage; Preferably, the renal tubular damage is caused by folic acid, adenine and / or unilateral ureteral ligation; preferably, the fibrosis epigenetic regulatory factors include one or more of α-sma, COL-1, COL-3, and E-cadherin.

10. The use according to claim 6, characterized in that: The drug also includes a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent, and a drug carrier with no toxic or side effects; preferably, the dosage form of the drug includes drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments, or creams.

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