Application of hempseed (-)-epicatechin-3-O-gallate

Abstract

The invention discloses application of hempseed (-)-epicatechin-3-O-gallate, and belongs to the technical field of biology. According to the invention, the EPI (-)-epicatechin-3-O-gallate extracted from the EPI is applied to the diabetic kidney diseases, so that the EPI can significantly reduce blood sugar and blood fat levels, improve pathological changes of the kidney of diabetic mice, delay senescence and fibrosis of renal tubular epithelial cells of the diabetic mice, improve the drug efficacy of DKD (Dikyl Ketene Dimer), and can be used for treating the diabetic kidney diseases. And reliable treatment targets and theoretical basis are provided for treating DKD by natural medicine monomer components.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of red sesame (-)-epicatechin-3-O-gallate in the preparation of a drug for improving renal tubular epithelial cell aging in diabetic nephropathy. Background Technology

[0002] Red hemp ( Lapotrea bulbifera (Sieb. et Zucc.) Wedd.) belongs to the genus *Artemisia* of the family Urticaceae. Laportea A plant. It commonly grows in moist, semi-shaded areas on mountain slopes or roadsides at altitudes of 1000-2400 meters. Also known as red hemp, wild green hemp, Ruida stick, or frog fight, it has long been used to treat rheumatoid arthritis and some other inflammations, such as itchy skin, fracture pain, and sports injuries. Crude extracts of red hemp are usually made into capsules or ointments, such as Runzao Zhiyang capsules, Liuwei Dihuang pills, and Compound Shangfuning ointment. Recent studies on the chemical composition of red hemp have revealed that its main chemical components are flavonoids, coumarins, steroids, and oils. Modern pharmacological studies have found that red hemp has significant anti-inflammatory, analgesic, immunosuppressive, hypoglycemic, lipid-lowering, and antiviral pharmacological effects.

[0003] Diabetic kidney disease (DKD) is one of the most serious complications of diabetes. The rate of kidney function loss in DKD is closely related to tubular injury and interstitial fibrosis. Renal tubular-interstitial fibrosis can show obvious tubular atrophy, epithelial-mesenchymal transition (EMT) and deposition of extra cellular matrix (ECM). Many studies have shown that the occurrence and development of DKD are closely related to cell aging. Inhibition of the development of senescent renal tubular epithelial cells in mice can significantly delay the progression of DKD. Therefore, exploring the mechanism and drugs to prevent or delay aging and related kidney diseases aggravated by aging has gradually become a research hotspot. The US Food and Drug Administration has approved four anti-aging drugs for diabetes: sodium-glucose co-transporter 2 (SGLT2) inhibitors, metformin, bisphosphonate drugs and glucagon-like peptide-1 (GLP-1) receptor agonists. (1) SGLT2 inhibitors mainly inhibit SGLT2 in the kidney, increase urinary glucose excretion and reduce blood glucose; reduce glomerular hyperfiltration and tubular glucose toxicity, delay the progression of diabetic nephropathy; and reduce kidney aging-related markers. (2) Metformin: Metformin can inhibit hepatic gluconeogenesis, improve peripheral insulin sensitivity, regulate intestinal function, reduce glycogenolysis, reduce pro-inflammatory and pro-fibrotic factors secreted by senescent cells, and delay tissue functional decline. Metformin can play an anti-aging role through multiple pathways such as mitochondrial regulation, anti-inflammation and enhanced autophagy. (3) Bisphosphonate drugs can reduce pro-inflammatory factors, inhibit the mevalonate pathway, reduce the survival of senescent cells and the release of inflammatory factors, affect telomerase activity or prevent telomere shortening, and delay the progression of cell aging. (4) GLP-1 receptor agonists: GLP-1 receptor agonists can stimulate insulin secretion, inhibit glucagon, improve insulin sensitivity and resist cell aging when blood glucose is elevated. The above four types of drugs are artificially synthesized drugs and are widely used in the treatment of diabetes and the delay of aging, but they also have non-negligible side effects. The side effects of SGLT2 inhibitors include: ① ketosis acidosis; ② the drug has osmotic diuresis, which may cause symptoms of volume depletion; ③ reversible mild elevation of serum creatinine, increased urinary calcium excretion, and mild elevation of low-density lipoprotein cholesterol; ④ increased risk of genital fungal infections, urinary tract infections and bladder dysfunction; ⑤ increased risk of bladder cancer. The side effects of metformin include gastrointestinal discomfort, vitamin B12 deficiency, lactic acidosis, hypoglycemia and skin rash.The side effects of bisphosphonate drugs are shown in fever, vomiting, skin rash, diarrhea, dizziness, abdominal pain, muscle and bone pain, headache, allergic reaction, chest pain, ulcerative stomatitis, hypocalcemia, palpitation, anorexia, indigestion, edema and other symptoms. The side effects of GLP-1 receptor agonists are shown in liver injury, gastrointestinal discomfort, allergy and the like.

[0004] Compared with artificial synthetic drugs, natural drug monomer components (such as single active compounds extracted from plants, microorganisms or animals) have the advantages of structural diversity, multi-target synergy, high safety and sustainable source, and natural products have unique chemical skeleton after long-term natural selection, can efficiently target biomolecules, many natural monomers can simultaneously regulate multiple pathways, meet the treatment needs of complex diseases, and have great potential in anti-aging and complex metabolic diseases. The natural source monomer is often more compatible with the biological system, and the side effects are lower than those of artificial synthetic drugs. Therefore, it is of great significance to use redweed natural extract for diabetic kidney disease. SUMMARY

[0005] In order to solve the above problems, the application provides a use of (-)-epicatechin-3-O-gallate in redweed in preparation of a drug for improving senescence of renal tubular epithelial cells in diabetic nephropathy. The extraction rate and purity of (-)-epicatechin-3-O-gallate can be improved, and the (-)-epicatechin-3-O-gallate in redweed is applied to preparation of a drug for improving senescence of renal tubular epithelial cells in diabetic nephropathy, so that the pathology of diabetic kidney can be improved, and the senescence and fibrosis of renal tubular epithelial cells in diabetic mice can be delayed.

[0006] The preparation method of the (-)-epicatechin-3-O-gallate in redweed comprises the following steps: (1) grinding redweed rhizomes, adding an organic solvent for extraction to obtain a crude extract; (2) dissolving the crude extract in pure water, then adsorbing and separating by using an adsorption resin column, eluting with an eluent, collecting elution fractions, removing the eluent by reduced pressure distillation, drying the obtained fractions to obtain an epicatechin gallate crude extract; (3) adsorbing and separating the epicatechin gallate crude extract again by using an adsorption resin, collecting elution fractions after elution with an eluent, drying, then recrystallizing, filtering, and freeze-drying the crystals to obtain a product, which is (-)-epicatechin-3-O-gallate (EPI) in redweed.

[0007] Further, the organic solvent in step (1) is ethyl acetate, the amount of the ethyl acetate is 8-10 times the weight of the redweed rhizomes, and the extraction conditions are as follows: reflux extraction at 35-70 DEG C for 4-12 h.

[0008] Further, the crude extract of step (2) is dissolved in pure water to prepare a column loading solution with a concentration of 3% (w / v); the elution solvent is a mixed solvent; the column loading flow rate is 1.0 BV / h; the elution is performed by first eluting the fraction rich in non-ester-type catechins with 2 BV of 25% ethanol, and then eluting the fraction rich in ester-type catechins with 2 BV of the mixed solvent, and the elution flow rate is 1.0 BV / h; the mixed solvent is a mixture of ethanol, methanol and isopropanol, and the volume ratio of ethanol, methanol and isopropanol in the mixed solvent is 10:2:1. The use of the mixed solvent can improve the extraction rate of (-)-epigallocatechin-3-O-gallate from Radix Rubus Suavissimi.

[0009] Further, in the adsorption of step (3), the dried epigallocatechin gallate crude extract obtained in step (2) is dissolved in pure water to prepare a column loading solution with a concentration of 3% (w / v), and the column loading flow rate is 1.0 BV / h; the elution solvent is ethanol, and the ethanol desorption fraction is collected, concentrated to an alcohol degree of <5 by high-efficiency external circulation, further concentrated by a reverse osmosis membrane, and then centrifugally spray-dried to obtain the epigallocatechin gallate crude extract.

[0010] Further, the epigallocatechin gallate is recrystallized as follows: the epigallocatechin gallate crude extract is dissolved in pure water to prepare a saturated solution, and then an organic solvent is added for recrystallization, and the organic solvent is a mixture of methanol, n-propanol and isopropanol, and the volume ratio of methanol, n-propanol and isopropanol in the organic solvent is 2:1:1. The use of the mixed organic solvent can improve the purity of (-)-epigallocatechin-3-O-gallate monomers from Radix Rubus Suavissimi.

[0011] Further, the structural formula of the (-)-epigallocatechin-3-O-gallate from Radix Rubus Suavissimi is as follows: .

[0012] The application of the (-)-epigallocatechin-3-O-gallate from Radix Rubus Suavissimi has the following beneficial effects: (1) The (-)-epigallocatechin-3-O-gallate (EPI) in the application is a flavonoid monomer component isolated and extracted from Radix Rubus Suavissimi for the first time, and the (-)-epigallocatechin-3-O-gallate (EPI) has a significant inhibitory effect on cyclooxygenase-2 and a good scavenging activity on 1,1-diphenyl-2-trinitrobenzene hydrazine free radicals.

[0013] (2) The extracted (-)-epicatechin-3-O-gallate (EPI) of the red hemp can significantly reduce the blood sugar and blood lipid levels, improve the pathological changes of the kidney of the diabetic mouse, delay the aging and fibrosis of the renal tubular epithelial cells of the diabetic mouse, and improve the drug efficacy of DKD, thereby providing a reliable treatment target and theoretical basis for the natural drug monomer component in the treatment of DKD. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0015] Figure 1 is the mRNA expression level of cyclin-dependent kinase inhibitor 1 (p21) and cyclin-dependent kinase inhibitor 2 (p16) in the kidney of 22.8-month-old and 8.9-month-old mice, and the β-galactosidase staining and semi-quantitative statistical diagram, wherein n=6, *p<0.05; Figure 2 is the expression of biochemical indicators of each group of mice after EPI treatment, wherein n=6, *p<0.05; Figure 3 is the pathological morphological change of each group of mice after EPI treatment; Figure 4 is the result graph of EPI reducing the mRNA expression of p21 in the renal tubular epithelial cells of the old diabetic mouse, wherein n=6, *p<0.05; Figure 5 is the result graph of EPI improving the aging proteins p21 and p16 in the renal tubular epithelial cells of the old diabetic mouse, wherein n=6, *p<0.05; Figure 6 is the result graph of EPI reducing the fluorescence expression and localization of p21 in the kidney tissue of the old diabetic mouse.

[0016] Figure 7 is the result graph of EPI reducing the contents of TNF-α and IL-6, which are the aging-related secretory phenotype indicators of the old diabetic mouse, wherein n=6, *p<0.05; Figure 8 is the result graph of EPI improving the renal fibrosis of the old diabetic mouse, wherein n=6, *p<0.05; Figure 9 Figure 2 is a graph of the immunohistochemical results of EPI reducing FN deposition in the kidney tissues of old diabetic mice. DETAILED DESCRIPTION

[0017] The application will be further described in detail below with reference to specific examples and drawings.

[0018] Example 1

[0019] A preparation method of redweed (-)-epicatechin-3-O-gallate, comprising the following steps: (1) grinding the rhizome of redweed, adding an organic solvent for extraction, the organic solvent being ethyl acetate, the amount of ethyl acetate being 10 times the weight of the redweed rhizome, and the extraction condition being reflux extraction at 70°C for 12 hours to obtain a crude extract; (2) dissolving the crude extract in pure water to prepare a 3% (w / v) column loading solution, and then performing adsorption separation on an adsorption resin column; the column loading flow rate being 1.0 BV / h; elution: first eluting a fraction rich in non-ester type catechins with 2 BV of 25% ethanol, and then eluting a fraction rich in ester type catechins with 2 BV of a mixed solvent to elute the epicatechin gallate crude extract, the elution flow rate being 1.0 BV / h; the mixed solvent being a mixture formed by ethanol, methanol and isopropanol in a volume ratio of 10:2:1; collecting the elution fractions, removing the elution solvent by reduced pressure distillation, drying the obtained fractions, and obtaining the epicatechin gallate crude extract; (3) performing adsorption separation on an adsorption resin column again with the epicatechin gallate crude extract; when the adsorption resin is adsorbed, first dissolving the dried epicatechin gallate crude extract obtained in step (2) in pure water to prepare a 3% (w / v) column loading solution, and then performing column loading at a flow rate of 1.0 BV / h; the elution solvent being ethanol; collecting the ethanol desorption fractions, and then performing high-efficiency external circulation to concentrate and recover the ethanol to an alcohol degree of <5, further concentrating by reverse osmosis membrane, and then performing centrifugal spray drying to obtain the epicatechin gallate crude extract. Then, dissolving the epicatechin gallate crude extract in pure water to form a saturated solution, adding an organic solvent formed by methanol, n-propanol and isopropanol in a volume ratio of 2:1:1, and then placing the mixture at room temperature to perform recrystallization. Filtering the crystals, and then freeze-drying the crystals to obtain the product, which is redweed (-)-epicatechin-3-O-gallate (EPI).

[0020] The structural formula of the redweed (-)-epicatechin-3-O-gallate (EPI) is as follows: .

[0021] Example 2 Use of a red vine (-)-epicatechin-3-O-gallate in the preparation of a drug for improving the senescence of renal tubular epithelial cells in diabetic nephropathy, specifically: I. Animal experiments

[0022] 1. Modeling principle Multiple low-dose streptozotocin (STZ) injections can destroy the stability of beta cells and autoimmunity, and high-fat diet combined with low-dose STZ can induce mice to form type 2 diabetes from insulin resistance to STZ-induced beta cell dysfunction.

[0023] 2. Experimental animals Male C57BL / 6J mice, body weight (22±2) g, clean level, animal room constant temperature and humidity, temperature 20-22℃, humidity 40-70%, light 12 hours light and dark alternation.

[0024] 3. Experimental materials (-)-epigallocatechin gallate (EPI) obtained in Example 1, streptozotocin (STZ), beta galactose staining kit, dimethyl sulfoxide (DMSO), PEG300, physiological saline, insulin needle, 8 gauge gavage needle, electronic balance, biochemical analyzer, 4. Experimental methods and steps 4.1 Animal grouping and treatment 4.1.1 Random grouping: ① 6-week-old C57BL / 6J mice were fed with nutrient granules, free to drink pure water, and the temperature and humidity were suitable. When the mice were 68 weeks old, the mice showed obvious signs of reduced voluntary activity, gray or brown back hair, thinning back hair, and cloudy eyes. The 68-week-old old mice were randomly divided into two groups: Old normal group (NC-old, 10): always fed with nutrient granules; The old model group (DM-old, 30) was fed with high-fat feed containing 60% fat for 16 weeks. The insulin resistance level of each group of mice was calculated. The insulin resistance index of the old normal group (NC-old) was 0.35, and the insulin resistance index of the old model group (DM-old) was 3.60. The insulin resistance index of the old model group (DM-old) was significantly higher than that of the old normal group (NC-old), indicating that the old model group (DM-old) mice successfully induced insulin resistance. Then the old model group (DM-old) was injected with streptozotocin (STZ) (55 mg / kg) for modeling, continuously for 5 days, of which 10 were fed with high-fat feed containing 60% fat for 14 weeks, and the other 20 model mice were fed with high-fat feed containing 60% fat for 12 weeks and then treated with EPI for 2 weeks. The old normal group (NC-old) and the old model group (DM-old) were sacrificed at 22.8 months of age.

[0025] ②In order to verify whether the 22.8-month-old old mice have obvious aging lesions: another 6-week-old C57BL / 6J mice were used as young controls. After 1 week of adaptive feeding, the mice were divided into 2 groups: Young normal group (NC-young, 10): always fed with nutrient granules; Young model group (DM-young, 10): the same as the old model group (DM-old), after 16 weeks of feeding with high-fat feed containing 60% fat, the fasting blood glucose and insulin levels of the young normal group (NC-young) and the young model group (DM-young) were measured. The experimental results showed that the insulin resistance index of the NC-young group was 0.15, and the insulin resistance index of the DM-young group was 3.43. The insulin resistance index of the DM-young group was significantly higher than that of the NC-young group, and the DM-young group was successfully induced to form insulin resistance. Then STZ injection (55 mg / kg) was used for modeling, continuously for 5 days. The DM-young group was modeled for the same period as the DM-old group. After 14 weeks of feeding with high-fat feed containing 60% fat, the NC-young group and the DM-young group were sacrificed at the same time. The mice were 8.9 months old.

[0026] 4.2 Drug preparation: (1) Preparation of EPI solution 50 mg of EPI powder was weighed and dissolved in DMSO (250 ul), PEG300 (250 ul) and physiological saline (2000 ul) in turn, and shaken uniformly.

[0027] (2) Dosing method: EPI low dose 50 mg / kg, high dose group 100 mg / kg; DM-old group was given intragastrically, 6 days a week, for 2 weeks. The mortality of EPI high dose group mice increased during the administration period. The analysis of the reason may be that the EPI dose exceeds the tolerance threshold, and the old mice are more sensitive to drug dose, so the experiment mainly uses the EPI low dose group.

[0028] 4.3 Experimental method 4.3.1 Mouse frozen section The tissue was placed in the frozen embedding agent, cooled and solidified at-20℃, then sectioned by a frozen section machine, 4µm. The cut tissue was immediately placed in 4% paraformaldehyde for 20 min, and then washed with PBS. β galactose staining was performed, and the specific formula is shown in Table 1: Incubate at 37℃ for one week. Observe the coloration under a microscope and take pictures for preservation.

[0029] 4.3.2 Mouse kidney paraffin section preparation (1) Tissue fixation: Take about 2 mm of kidney tissue and put it into the embedding box. Fix it with 4% paraformaldehyde for 48 h, and slowly rinse it with running water for 2 h.

[0030] (2) Tissue dehydration: Perform gradient ethanol dehydration from low concentration to high concentration, 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, each for 40 min, 100% ethanol I, 100% ethanol II, each for 60 min, ethanol and xylene 1:1 mixture for 30 min, xylene I for 20 min, xylene II for 15 min, paraffin I and II at 65℃ for 60 min.

[0031] (3) Tissue embedding: Select the appropriate embedding mold, put the tissue in the middle of the mold, quickly drop the paraffin, then place it on the cooling table to fix the tissue. Take out the mold when the paraffin is completely solidified.

[0032] (4) Tissue sectioning: Label the corresponding information on the adhesive glass slide, use a paraffin sectioning machine to cut 3µm tissue, use the labeled glass slide to lift it after spreading, dry the water, and then put it into the section box for standby.

[0033] 4.3.3 HE staining, PAS staining, Sirus red staining (1) Put the tissue section into a 70℃ oven for 2 h for deparaffinization (2) Put it into xylene I for 5 min, xylene II for 5 min, xylene III for 5 min, anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, 95% ethanol for 1 min, 75% ethanol for 1 min, and rinse with running water for 15 min.

[0034] (3) Eosin staining for 2 min, PAS reagent staining for 2 min, Sirus red staining for 30 min, hematoxylin staining for 5-10 min (determined by observation under a microscope), water washing, 1% hydrochloric acid ethanol differentiation for about 3 s, water flushing for 10 min, eosin staining for 1 min.

[0035] (4) Dehydration and mounting: sequentially placing the section into 75% ethanol for 3 s, 95% ethanol for 3 s, anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, xylene for 2 min, and neutral resin mounting.

[0036] 4.3.4 Kidney immunohistochemical staining (1) Placing the tissue section into a 70°C oven for 2 h for deparaffination (2) Sequentially placing into xylene I for 5 min, xylene II for 5 min, xylene III for 5 min, anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, 95% ethanol for 1 min, 75% ethanol for 1 min, and water flushing for 15 min.

[0037] (3) The antigen repair process adopts a hot-induced epitope repair method. 1.0 mol / L sodium citrate buffer solution (pH 6.0) is injected into the staining rack containing the slide, and then heated to boiling by microwave, and then taken out and naturally cooled at room temperature. Then the sample is washed with PBS three times, each for 5 minutes.

[0038] (4) To eliminate endogenous peroxidase activity, evenly cover the tissue section surface with 3% hydrogen peroxide blocking agent, and incubate in a 37°C incubator for 30 minutes, and then wash with PBS three times, each for 5 minutes.

[0039] (5) In the primary antibody reaction stage, the FN (1:100) primary antibody working solution is added dropwise to the sample surface, and then transferred to a wet box, and incubated overnight at 4°C.

[0040] (6) The next day, the sample is restored to room temperature for 30 minutes, and then washed with PBS three times, each for 5 minutes.

[0041] (7) The secondary antibody incubation adopts HRP-labeled goat anti-rabbit / mouse IgG polymer (kit matching), and reacts at room temperature for 60 minutes, and then washed with PBS three times, each for 5 minutes.

[0042] (8) The DAB color development reaction is performed under the monitoring of an optical microscope, and when the target area presents an appropriate staining intensity, immediately terminate the reaction with deionized water.

[0043] (9) The cell nucleus staining adopts Harris hematoxylin solution, and the staining time is controlled at 30 seconds, and the staining effect is observed in real time.

[0044] (10) After dehydration by gradient ethanol and clearing by xylene, the sections were observed and images were collected under an optical microscope after being fixed by neutral gum.

[0045] 4.3.5 Immunofluorescence staining of tissues (1) The kidney tissues were prepared into 5-μm-thick frozen sections. The sections were fixed in 4% paraformaldehyde solution at room temperature for 20 minutes, and then transferred to a staining rack and washed with phosphate buffered saline (PBS) three times for 5 minutes each time on a horizontal shaker.

[0046] (2) 50 μL of 0.5% Triton X-100 permeabilizing agent was added to each section to enhance cell membrane permeability. After treatment, the sections were again washed with PBS three times on a horizontal shaker, with an interval of 5 minutes each time.

[0047] (3) To reduce non-specific binding, 100 μL of 5% bovine serum albumin (BSA) blocking solution was evenly coated on the surface of the tissues, and incubated in a 37°C incubator for 30 minutes. After removing the blocking solution, p21 primary antibody (1:50) diluted with 1% BSA was added, and the wet box was incubated at 4°C overnight.

[0048] (4) The sample was equilibrated at room temperature for 30 minutes, and washed with PBS three times for 5 minutes each time. Then 50 μl of Cy3-labeled goat anti-rabbit IgG secondary antibody was added, and incubated at room temperature for 1 hour in the dark. After the reaction was completed, the sections were washed with PBS three times for 5 minutes each time.

[0049] (5) The sections were mounted using a fluorescent mounting medium containing DAPI, and the entire process was performed in the dark. After the sample preparation was completed, the laser confocal microscope was used to observe and collect fluorescence images.

[0050] 4.3.6 Real-time fluorescent quantitative PCR (1) 100 mg of kidney tissue sample was accurately weighed and added to 1 mL of Trizol lysis solution. The tissue homogenizer was used to break the sample at a frequency of 50 Hz for 1 minute. The lysis solution was transferred to a 1.5 mL centrifuge tube, and centrifuged at 12000 x g at 4°C for 20 minutes. The supernatant was collected into a new centrifuge tube.

[0051] (2) 200 μL of chloroform was added to the centrifuge tube containing the supernatant, and vortexed for 30 seconds. The solution was allowed to stand at room temperature for 15 minutes. Then the solution was centrifuged at 12000 x g at 4°C for 15 minutes to separate the layers.

[0052] (3) After centrifugation, the upper aqueous phase was carefully aspirated using an RNase-free pipette tip, and transferred to a new 1.5 mL RNase-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was gently mixed and allowed to stand at room temperature for 10 minutes.

[0053] (4) Centrifuge the sample at 12000 x g for 20 minutes at 4°C, discard the supernatant, and keep the RNA pellet at the bottom of the tube.

[0054] (5) Add 500 μΐ of pre-cooled 75% ethanol to the centrifuge tube containing the RNA pellet, gently mix, and centrifuge at 12000 x g for 5 minutes at 4°C. Discard the supernatant.

[0055] (6) Repeat the ethanol wash step once, and then invert the centrifuge tube on sterile filter paper and air dry for 15 minutes at room temperature.

[0056] (7) Dissolve the RNA pellet with an appropriate amount of DEPC water, and use a microspectrophotometer to determine the RNA concentration and purity (A260 / A280 ratio). After aliquoting, store at -80°C in an ultra-low temperature freezer.

[0057] (8) Refer to the Hifair® III First-Strand cDNA Synthesis Kit (with gDNA Removal Enzyme) instruction manual to prepare the reaction system. Gently mix the mixture, and then transfer to a 42°C metal bath for heating for 2 minutes to complete the genomic.

[0058] (9) Add 4x Hifair® III SuperMix plus reaction mixture to the above reaction solution, gently mix, and immediately perform the reverse transcription reaction.

[0059] (10) The experimental operation strictly follows the standardized process of the Hieff UNICON® Universal SYBR Green qPCR Master Mix. Refer to the recommended protocol in the instruction manual for the preparation of the reaction system, and all steps are operated on ice to ensure the stability of the reagents. After the system is configured, add the eight-tube tube according to the amount, and perform the operation.

[0060]

[0061] 4.3.7 Western blot detection (1) Preparation of animal tissue protein samples ① Preparation: a set of sterile and enzyme-free forceps, scissors, three sets of 1.5 ml test tubes, and a set of 2 ml round-bottom test tubes each containing 4 sterile and enzyme-free homogenization beads, open the 4°C centrifuge in advance to cool, and take the mouse kidney tissue from the liquid nitrogen freezer and place it in crushed ice.

[0062] ② Weigh the tissue: use sterile and enzyme-free forceps and scissors to weigh the kidney tissue on ice, 10 mg per mouse, then place it in the 2 ml test tube containing the homogenization beads prepared in advance, and store it on ice. After weighing all the tissues, add 260 μΐ of protein lysis solution to each tube, and immediately store the remaining tissue in the liquid nitrogen freezer to prevent degradation. ​

[0063] ③ Tissue homogenate: Put the weighed tissue into the tissue grinder, grind for 60 seconds, 60 Hz, and repeat three times. After each grinding, perform a brief centrifugation.

[0064] ④ Take the protein supernatant: Put the homogenized sample into a pre-cooled 4°C centrifuge at 12000 rpm for 15 minutes. Then, take the supernatant and transfer it into a 1.5 ml test tube prepared in advance, and immediately insert it into ice.

[0065] ⑤ BCA assay: Take a new 1.5 ml test tube and dilute the protein stock solution ten times (18 ul distilled water + 2 ul protein stock solution). Take a 96-well plate, add the standard and diluted sample to each well (10 ul per well), and add 200 ul of BCA working solution (prepared fresh) to each well. Gently mix, incubate in a 37°C incubator for 30 minutes, and use a microplate reader to measure the absorbance at a wavelength of 562 nm.

[0066] ⑥ Protein concentration calculation: Calculate the standard protein concentration and corresponding absorbance to obtain a scatter plot with protein concentration ug / ul as the horizontal coordinate and absorbance as the vertical coordinate. Add a trend line, display the formula and R2 (R2 > 0.99 is required), and draw a standard curve. Enter the absorbance of each sample to calculate its protein concentration.

[0067] ⑦ Preparation of loading buffer: Calculate the required volumes of protein stock solution, ddH2O, and 5x loading buffer based on a protein concentration of 3 ug / ul. Mix them in a new 1.5 ml test tube and heat in a 100°C metal bath for 10 minutes to obtain the loading buffer. Then, proceed with the subsequent electrophoresis or store it in a -20°C refrigerator.

[0068] (2) Preparation of PAGE gel

[0069] Prepare the separation gel according to Table 3, mix immediately, and add to the glass plate at about five quarters of the position. Gently add distilled water to flatten, and let stand for 30 minutes. After the water and gel separate, pour out the water, wipe off the remaining water, prepare the concentrated gel, pour it into the glass plate immediately, and insert a 1.5 mm comb. After 20 minutes, remove the comb.

[0070] (3) Separate proteins ① Loading: Place the gel plate and electrophoresis frame into the electrophoresis tank and fix them in the corresponding grooves. Pour in the electrophoresis buffer 1X loading buffer. Take out the stored loading buffer from -20°C and thaw it at room temperature. Use a pipette or loading needle to slowly load 5-10 ul of sample liquid, and note that 2.5 ul of protein marker should be added to both ends of the sample.

[0071] ②Electrophoresis: two wires are connected to the electrophoresis tank and the electrophoresis apparatus, pay attention to the positive and negative poles of the wire. First, adjust the voltage to 80V, when the red marker appears, generally for 1 hour, then adjust the voltage to 120V, continue electrophoresis for 1 hour or so, the blue indicator reaches the low end, stop electrophoresis.

[0072] (4) Transfer film ① Configure the transfer buffer 1X Transfer Buffer, soak the PVDF film in methanol for 1 min, prepare a plate and pour the transfer buffer.

[0073] ② Open the transfer film device, black down, put in sponge, two filter paper, glue, activated PVDF film, two filter paper, sponge, close the transfer film device, put into the electric transfer tank, add electric transfer liquid, plug in the wire, pay attention to the positive and negative poles. The whole electrophoresis device is placed in a basin and ice is added for ice bath. Adjust the voltage to 90V, transfer film for about 2 hours.

[0074] (5) Blocking ① Configure 5% skim milk, soak the film in it and place it on the shaker at room temperature for 1 hour.

[0075] ② After blocking, according to the protein molecular weight, trim the corresponding band, and then wash with TBST for 3 times, 7 min each time.

[0076] (6) Primary antibody incubation ① Configure the diluted primary antibody (Fibronectin, E-cadherin, Collagen-III, E-cadherin, a-SMA, p21, p16, β-catenin), immediately add the primary antibody after the TBST in the box is absorbed, and incubate in a 4°C shaker overnight.

[0077] ② After incubation, recover the primary antibody, and wash the film with TBST for 3 times, 7 min each time.

[0078] (7) Secondary antibody incubation ① Configure the diluted secondary antibody (horseradish peroxidase labeled goat anti-rabbit IgG), immediately add the secondary antibody after the TBST in the box is absorbed, and incubate on the shaker at room temperature for 1 hour.

[0079] ② After incubation, recover the secondary antibody, and wash the film with TBST for 3 times, 7 min each time.

[0080] (8) Exposure Configure the chemiluminescence solution (A:B=1:1) and avoid light. Take the film soaked in TBST, forceps, paper towel, and pipette into the exposure room, turn on the machine, absorb the remaining TBST on the film with paper towel, then add the chemiluminescence solution for exposure.

[0081] 4.3.8 ELISA assay After blood is static for half an hour, the blood sample is then centrifuged for 15 min at 3000 r / min, and the separated serum is aliquoted into sterile 1.5 ml centrifuge tubes and stored in a -80℃ ultra-low temperature refrigerator for standby. The separated serum is used to measure the expression contents of the biochemical indexes and the mouse aging-related secretory phenotype indexes, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6).

[0082] (1) The antigen or antibody is diluted to a working concentration (5 μg / mL) with a coating buffer (such as a carbonate buffer, pH 9.6), 100 μL per well of a 96-well plate, and incubated at 4℃ overnight (or 37℃ for 2 hours).

[0083] (2) Discard the liquid in the wells, wash the plate 3 times with a washing buffer (PBS-T), soak for 2 minutes each time, and pat dry.

[0084] (3) Add 300 μL of blocking solution to each well, and incubate at 37℃ for 2 hours. After blocking, wash 3 times.

[0085] (4) Add the diluted serum sample to be tested, 100 μL per well, and set duplicate wells. At the same time, set blank wells (only buffer), negative controls and positive controls, incubate at 37℃ for 2 hours, and wash 3 times.

[0086] (5) Add an enzyme-labeled secondary antibody, incubate at 37℃ for 1 hour, and wash 5 times.

[0087] (6) Add a color developing substrate, 100 μL per well, and incubate at room temperature for 30 minutes in the dark.

[0088] (7) Add 50 μL of a stopping solution to each well, immediately detect the OD value with an enzyme label instrument, and calculate the concentration of the sample to be tested according to the standard curve.

[0089] The expression of biochemical indexes of mice in each group after EPI treatment, pathological staining and PCR test results are shown in Figures 1-4 ; The QPCR results of Figure 1 show that the expression levels of p21 and p16 genes in the kidneys of the DM-old group are significantly higher than those in the NC-old group and the DM-young group. The β-galactosidase staining results show that the blue staining positive of the kidneys of the DM-old group is significantly more than that of the NC-old group and the DM-young group, indicating that the 22.8-month-old old diabetic mice have obvious aging characteristics, which provides a strong animal model for exploring the improvement of DKD renal tubular epithelial cell aging by EPI.

[0090] The QPCR results of Figure 2Biochemical index results showed that compared with the NC-old group, the urea nitrogen (Blood ureanitrogen, BUN), creatinine (Serum creatinine, Scr), urine microalbumin (Microalbumin, MAU) of DM-old group mice were significantly increased, and the EPI treatment group was significantly reduced, indicating that EPI can effectively improve the damage of kidney function of DM-old mice; The blood glucose (Blood glucose, BG) of DM-old group mice was significantly higher than that of NC-old group mice, and the BG of mice after EPI treatment was significantly lower than that of DM-old group, indicating that EPI can effectively reduce the blood glucose level of DM-old mice; The serum total cholesterol (Totalcholesterol, T-CHO) and triglyceride (Triglyceride, TG) of DM-old group mice were significantly higher than those of NC-old group, and the T-CHO and TG levels of EPI group were significantly reduced, which indicated that EPI can alleviate the lipid metabolism disorder of DM-old mice.

[0091] Figure 3 The pathological morphological changes of each group of mice after EPI treatment. HE staining and PAS staining results showed that the kidney structure of NC-old group mice was clear and complete, the basement membrane was not significantly thickened, and the renal tubules were arranged in order; The shedding of renal tubular epithelial cells in DM-old group caused local atrophy of renal tubules, and lymphocyte infiltration in renal interstitium; Sirus red staining results showed that there was obvious pink collagen fiber deposition in the renal interstitium of DM-old group. The degree of the above-mentioned lesions and collagen fiber deposition in EPI group were improved compared with DM-old group.

[0092] Figure 4 The results of EPI reducing the expression of senescence gene p21 mRNA in renal tubular epithelial cells of old diabetic mice. QPCR results showed that compared with NC-old group, the p21 mRNA level in the renal cortex of DM-old mice was significantly increased, and after EPI treatment, the mRNA level of p21 was significantly decreased.

[0093] Western blot and immunofluorescence test results: As shown in Figure 5 , compared with the NC-old group, the expression of p21 and p16 proteins in the kidney of DM-old group mice increased, and the expression of p21 and p16 proteins significantly decreased after EPI treatment, indicating that EPI can improve the senescence proteins p21 and p16 of renal tubular epithelial cells in old diabetic mice. Immunofluorescence as Figure 6 shown, the p21 fluorescence expression of renal tubular epithelial cells in DM-old group was significantly more than that in NC-old group, which was located in the nucleus, and the p21 fluorescence signal was significantly weakened after EPI treatment.

[0094] As can be seen from Figure 8 , compared with the NC-old group, the expression of fibronectin (FN), alpha smooth muscle actin (a-SMA), type III collagen (Col-III) proteins in the kidney of the DM-old group mice increased, and the expression of E-cadherin protein decreased, and after EPI treatment, the expression of FN, a-SMA and Col-III proteins decreased significantly, and the expression of E-cadherin protein increased.

[0095] Serum ELISA detection results: As Figure 7 shown, compared with the NC-old group, the expression of aging-related secretory phenotype indicators TNF-α and IL-6 in the serum of the DM-old group mice increased significantly, and after EPI treatment, the levels of TNF-α and IL-6 decreased significantly.

[0096] Figure 9 The immunohistochemical results of EPI reducing FN deposition in the kidney tissue of old diabetic mice are shown in the figure. The immunohistochemical results show that the FN brownish yellow linear positive expression in the kidney of the DM-old group is obviously more than that in the NC-old group, and after EPI treatment, the FN brownish yellow staining expression decreases significantly.

[0097] The above describes the technical solutions provided by the embodiments of the present application in detail. The principles and implementation modes of the embodiments of the present application are described by applying specific examples, and the above description of the embodiments is only applicable to help understand the principles of the embodiments of the present application; at the same time, for those skilled in the art, according to the embodiments of the present application, the specific implementation modes and application scope will be changed, and the above description of the embodiments should not be understood as a limitation of the present application.

Claims

1. Use of (-)-epigallocatechin-3-O-gallate in preparing a medicine for improving the senescence of renal tubular epithelial cells in diabetic nephropathy.

2. The use of red vine (-)-epicatechin-3-O-gallate according to claim 1 for the preparation of a medicament for improving the senescence of renal tubular epithelial cells in diabetic nephropathy, characterized in that, The structure of the (-)-epigallocatechin-3-O-gallate is as follows: 。 3. The use of red vine (-)-epicatechin-3-O-gallate in the preparation of a drug for improving the senescence of renal tubular epithelial cells in diabetic nephropathy according to claim 1, characterized in that, The preparation method of the (-)-epigallocatechin-3-O-gallate comprises the following steps: (1) grinding the rhizome of Rubus corchorifolius, adding an organic solvent to extract to obtain a crude extract; (2) dissolving the crude extract with pure water, then adsorbing and separating with an adsorption resin column, eluting with an eluent, collecting the elution fraction, removing the eluent by reduced pressure distillation, drying the fraction to obtain a crude epigallocatechin gallate extract; (3) adsorbing and separating the crude epigallocatechin gallate extract again with an adsorption resin, collecting the elution fraction after eluting with an eluent, drying, recrystallizing, filtering, and freeze-drying the crystals to obtain the product, which is the (-)-epigallocatechin-3-O-gallate.

4. The use of red vine (-)-epicatechin-3-O-gallate in the preparation of a drug for improving the senescence of renal tubular epithelial cells in diabetic nephropathy according to claim 2, characterized in that: In step (1), the organic solvent is ethyl acetate, the amount of the ethyl acetate is 8-10 times the weight of the rhizome of Rubus corchorifolius, and the extraction conditions are as follows: reflux extraction at 35-70°C for 4-12 h.

5. The use of red vine (-)-epicatechin-3-O-gallate according to claim 4 for the preparation of a medicament for improving the senescence of renal tubular epithelial cells in diabetic nephropathy, characterized in that: In step (2), the crude extract is dissolved with pure water to prepare a column feed solution with a concentration of 3% (w / v); the elution solvent is a mixed solvent; the mixed solvent is a mixture of ethanol, methanol and isopropanol, and the volume ratio of ethanol, methanol and isopropanol in the mixed solvent is 10:2:

1.

6. The use of red vine (-)-epicatechin-3-O-gallate according to claim 3 for the preparation of a medicament for improving the senescence of renal tubular epithelial cells in diabetic nephropathy, characterized by: In step (3), the eluent is ethanol.

7. The use of red vine (-)-epicatechin-3-O-gallate according to claim 3 for the preparation of a medicament for improving the senescence of renal tubular epithelial cells in diabetic nephropathy, characterized by: In step (3), the recrystallization is as follows: dissolving the crude epigallocatechin gallate extract with pure water to obtain a saturated solution, and then adding an organic solvent for recrystallization; the organic solvent is a mixture of methanol, n-propanol and isopropanol, and the volume ratio of methanol, n-propanol and isopropanol in the organic solvent is 2:1:1.

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