New application of ENPEP protein

Abstract

The invention discloses a novel application of ENPEP protein, and relates to the technical field of biology. The ENPEP protein can be used for resisting aging through overexpression. It is found for the first time that the expression level of ENPEP in senescence blood vessels is remarkably reduced, by changing the expression quantity of ENPEP, detecting protein expression of ENPEP and senescence-related markers and constructing senescence models of different VSMCs at the same time, the credibility of experimental results is greatly improved, a certain experimental basis and method are provided for treating senescence-related diseases, and the application prospect is wide. Experimental results of the invention prove that the overexpression of ENPEP can down-regulate the expression of aging-related marker genes p53, p21 and p16, and ENPEP can be used for preparing anti-aging drugs for prevention or treatment.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to a new use of ENPEP protein. Background Art

[0002] Glutamyl aminopeptidase (ENPEP) is a key gene regulating hypertension. It primarily regulates blood pressure by cleaving the N-terminal acidic residue of angiotensin II (Angll) through calcium regulation, converting Ang II to angiotensin III (Ang III). Subsequent studies have shown that ENPEP also influences angiogenesis. Furthermore, ENPEP plays a crucial role in cancer, diabetes, Parkinson's disease, immune function, and mitral valve prolapse. However, its role in aging has not been reported. Summary of the Invention

[0003] The present invention provides a new use of ENPEP protein, which aims to solve the problems existing in the above-mentioned background technology.

[0004] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions: In a first aspect, the present invention discloses a use of a preparation overexpressing ENPEP protein in anti-aging.

[0005] In a preferred embodiment of the present invention, the ENPEP protein has an amino acid sequence as shown in SEQ ID NO.1.

[0006] In a preferred embodiment of the present invention, the preparation overexpressing ENPEP protein achieves anti-aging effects by inhibiting the expression of p16, p21, and p53 aging-related marker genes.

[0007] In a second aspect, the present invention discloses the use of a reagent for specifically detecting the expression level of a gene used to translate ENPEP protein in the preparation of an anti-aging product.

[0008] In a preferred embodiment of the present invention, the product comprises a real-time fluorescence quantitative PCR kit.

[0009] In a preferred embodiment of the present invention, the kit contains a primer pair as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0010] In a preferred embodiment of the present invention, the PCR reaction system is 20 μL, and the components in the system are: PCRMix 10 μL, 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, 7 μL of deionized water, and 1 μL of cDNA; The reaction procedure is 10 min pre-denaturation at 95℃, 10 s denaturation at 95℃, 30 s annealing at 60℃, and a total of 40 cycles.

[0011] In a third aspect, the present application discloses a method for anti-aging of cells, wherein the ENPEP protein is transfected into cells to construct anti-aging cells overexpressing the ENPEP protein.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application firstly finds that the expression level of ENPEP in aging blood vessels is significantly reduced, and by changing the expression amount of ENPEP, the protein expression of ENPEP and aging-related markers is detected, and different VSMC aging models are constructed, which greatly improves the credibility of experimental results, and provides certain experimental basis and method for treating diseases related to aging, and the experimental results of the present application prove that overexpression of ENPEP can down-regulate the expression of aging-related marker genes p53, p21 and p16, and can be used for preparing anti-aging drugs. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure 1 is a diagram of the effect of ENPEP on aging, wherein A. ENPEP is found to be significantly down-regulated in aging intestinal glial cells in human aging data; B. ENPEP is found to be significantly down-regulated in aging renal proximal tubular epithelial cells in human aging data; C. KEGG analysis of differential proteins in proteomics after knockdown of ENPEP; Figure 2 Figure 2 is a diagram of down-regulation of ENPEP expression in aging blood vessels, wherein A. Immunohistochemistry and immunofluorescence are used to detect the expression and localization of ENPEP; B. Western blot is used to detect the protein expression of ENPEP and aging-related markers; C. qRT-PCR is used to detect the mRNA levels of ENPEP, p53, p21 and p16; D. ElISA is used to detect the contents of angiotensin Ⅲ and angiotensin Ⅱ in serum; Figure 3Figure 1 shows the downregulation of ENPEP expression in VSMCs of different aging models, including: H2O2-induced VSMCs aging model; AB. qRT-PCR and Western blot detection of ENPEP, p53, p21, and p16 expressions; C. β-galactosidase staining detection of senescent cell number; D. Elisa detection of angiotensin II and III in VSMCs and culture medium; Angiotensin II-induced VSMCs aging model; EF. qRT-PCR and Western blot detection of ENPEP, p53, p21, and p16 expressions; G. β-galactosidase staining detection of senescent cell number; H. Elisa detection of angiotensin II and III in VSMCs and culture medium; D-galactose-induced VSMCs aging model; IJ. RT-PCR and Western blot detection of ENPEP, p53, p21, and p16 expressions; K. β-galactosidase staining detection of senescent cell number; L. ELISA was used to detect the changes of angiotensin II and III in VSMCs and culture medium respectively; Figure 4 Figure 4 shows the expression of aging-related genes regulated by ENPEP. AB. After ENPEP knockdown, the expressions of ENPEP, p53, p21, and p16 were detected by qRT-PCR and Western blot. CD. After ENPEP overexpression, the expressions of ENPEP, p53, p21, and p16 were detected by qRT-PCR and Western blot. E. ELISA was used to detect the changes of angiotensin II and angiotensin III in VSMCs and culture medium, respectively. DETAILED DESCRIPTION

[0014] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0015] Example 1 1. Search for aging-related DEGs in the single-cell RNA of the human aging database AgeAnno, and search for ENPEP protein.

[0016] The results showed that ENPEP expression was significantly downregulated in aging small intestine and kidney tissues. Figure 1 As shown in A and B.

[0017] 2. To further explore the mechanism of action of ENPEP in the aging process, the applicant used siRNA interference technology to specifically knock down ENPEP expression in VSMCs, followed by proteomic analysis.

[0018] Specifically, in the vascular smooth muscle transfected with small interfering control (si-NC) and small interfering ENPEP (si-ENPEP); The transfection method is as follows: 1) According to the operation instruction of Lipofectamine® 2000 Reagent. Transfect when the cells in the six-well plate grow to 70-80%; 2) Add small interfering 8ul and Lipo 2000 8ul into double-free medium respectively, mix, centrifuge and stand at room temperature for 5 min, then add the double-free medium (ordinary DMEM medium) containing Lipo 2000 into the medium containing plasmid (the plasmid is pcDNA3.1-control and pcDNA3.1-ENPEP purchased from Hanheng Biological), mix, centrifuge and stand at room temperature for 20 min; 3) Wash the cells with PBS twice, add double-free medium, and then add the prepared mixture into the cell bottle and mix; 4) After transfection for 4 h, replace the medium with complete medium (double-free medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) and continue to culture for 24 h. After the end, send for transcriptome sequencing.

[0019] 3, use the differential gene to carry out KEGG enrichment analysis. The results are shown in Figure 1 Figure C, the KEGG pathway enrichment analysis results show that the differentially expressed proteins are significantly enriched in multiple aging-related signal pathways, which suggests that ENPEP may regulate the aging process by regulating these key pathways.

[0020] Example 2: The expression and distribution of ENPEP in aging vascular tissue were detected by immunohistochemistry and immunofluorescence techniques.

[0021] After paraffin sections were prepared from the aortas of 8-week-old and 21-month-old mice, immunohistochemical and immunofluorescent staining were performed. Then, the expressions of ENPEP, p16, p21 and p53 were detected by WB and PCR.

[0022] The method is as follows: 1. Tissue sampling: Anesthetize the mice, expose the chest and abdomen, heart and liver, and pre-cool PBS from the left ventricle to the aorta. The tissue around the aorta was stripped clean. The aorta was isolated.

[0023] 2. Paraffin embedding and sectioning: (1) Rinse with PBS and fix in 4% paraformaldehyde for 48 h.

[0024] (2) Wrap the vascular tissue with lens paper and rinse under running water for 10-15 min.

[0025] (3) Dehydration: 60% ethanol → 70% ethanol → 80% ethanol → 90% ethanol → anhydrous ethanol I (first anhydrous ethanol) → anhydrous ethanol II (second anhydrous ethanol) → anhydrous ethanol III (third anhydrous ethanol), 5 min each.

[0026] (4) Transparency: Transparency agent I (first transparent agent) → Transparency agent II (second transparent agent) → Transparency agent III (third transparent agent), each for 10 minutes. The trade name of the transparent agent is environmentally friendly transparent agent.

[0027] (5) Wax embedding: Wax the tissue block for 2 hours. Place the blood vessels vertically in an embedding box and place them in paraffin. Cool until solidified. Store the wax block at 4°C.

[0028] (6) Paraffin sections: Use a paraffin slicer to slice the slices to a thickness of 4 μm. Flatten the slices in water and place them on the broken pieces. Mark them. Bake the slices in an oven at 65°C for 1 hour. They can be used for subsequent experiments. If not used immediately, store them at 4°C.

[0029] 3. Dewaxing of sections: Take out the paraffin sections and bake them at 60℃ for 30min.

[0030] (1) Dewaxing: Clearing agent III → Clearing agent II → Clearing agent I, 10 minutes each.

[0031] (2) Hydration: Anhydrous ethanol III → Anhydrous ethanol II → Anhydrous ethanol I → 90% ethanol → 80% ethanol → 70% ethanol for 5 minutes each. Rinse slowly with running water for 5 minutes.

[0032] 4. Immunohistochemical staining: After dewaxing, perform antigen retrieval in a pressure cooker using citrate buffer. Place the sections in a retrieval box containing antigen retrieval solution and place in a pressure cooker. Heat the retrieval solution to 100°C for 3 minutes. Remove the retrieval box and cool at room temperature for 30 minutes. Rinse with PBS for 5 minutes three times. (1) Use a histochemical pen to draw a circle around the tissue. Use absorbent paper to remove the water around the tissue on the slice to avoid rubbing the tissue. Add 3% hydrogen peroxide to the circle and incubate at room temperature for 10 minutes. Rinse with PBS for 5 minutes, three times. Use absorbent paper to remove the water around the tissue on the slice to avoid diluting the working solution. (2) Add goat serum working solution for blocking and incubate at room temperature for 30 minutes; prepare the primary antibody and use antibody diluent as the primary antibody diluent.

[0033] (3) Pour off the blocking solution on the surface of the slice (do not wash), add the primary antibody to cover the entire tissue block, place the slice with the primary antibody in a humidified box, and refrigerator at 4°C overnight; (4) Incubate the sections overnight with the primary antibody at room temperature for 30 minutes. Rinse with PBS three times, 5 minutes each time. (5) Add secondary antibody (goat anti-rabbit IgG), incubate at 37°C for 30 min, and rinse with PBS three times, 5 min each time; (6) Add horseradish enzyme-labeled proteasome working solution, incubate at 37°C for 30 min, and rinse with PBS three times, each time for 5 min; (7) DAB (diaminobenzidine) color development: Add DAB color development solution to the tissue and observe the color development results under a microscope. When specific staining appears in the positive area and the background is not stained, place the slide in tap water to stop the color development; (8) Place the slide in hematoxylin for 10 seconds and rinse with tap water for 5 minutes, three times; (9) Differentiation with 1% hydrochloric acid and alcohol for 3 seconds, and then rinse with tap water for 5 minutes, 3 times; (10) Soak in 70%, 80%, 95% alcohol II, 95% alcohol I, and 100% alcohol II for 1 second each; 100% alcohol I, xylene III, xylene II, and xylene I for 2 minutes each; (11) Seal the slide with neutral gum; after the gum dries, observe under an electron microscope.

[0034] 5. Immunofluorescence: After dewaxing, perform antigen retrieval in a pressure cooker: Use citrate buffer as the retrieval solution for brain tissue sections. Place the sections in an antigen retrieval box containing antigen retrieval solution and place in a pressure cooker. Heat the retrieval solution to 100°C for 3 minutes. Remove from the retrieval box and cool at room temperature for 30 minutes. Rinse with PBS for 5 minutes, three times. (1) Use a histochemical pen to draw circles around the tissue and absorb the water around the tissue on the slice with absorbent paper to avoid wiping off the tissue. Rinse with PBS for 5 minutes, 3 times; (2) Remove the water around the tissue on the slice with absorbent paper, add goat serum to block, and incubate at room temperature for 30 minutes; prepare the primary antibody, the amount of primary antibody should cover the entire tissue block, and place the slice with the primary antibody in a light-proof humid box at 4°C refrigerator overnight; (3) Place the sections with the primary antibody overnight in a dark place and incubate at room temperature for 30 minutes; rinse with PBS for 5 minutes, 3 times; and rinse with 0.5% Triton-PBS for 5 minutes, 3 times; (4) Add secondary antibody and incubate at 37°C for 1 h; (5) After incubation, rinse with PBS for 5 minutes, three times; (6) DAPI coverslips.

[0035] The results show that Figure 2Figure 6A shows that the expression level of ENPEP in aged vascular tissue is significantly reduced compared with young tissue, and this down-regulation mainly occurs in VSMCs.

[0036] Example 3 The expression of ENPEP was detected at the protein and mRNA levels by Western blot and RT-PCR techniques, respectively. The method is as follows: Polyacrylamide gel electrophoresis: The tissues were homogenized in RIPA buffer supplemented with protease inhibitors. The protein content was standardized using the BCA assay. The protein lysate (30 μg) in the cell sample was separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated with a primary antibody specific for the target protein, including ENPEP, p16, p21 and β-actin, at 4°C overnight. Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit) for 1 hour. The expression level of the protein was analyzed.

[0037] RNA extraction 1) Add 1 mL of RNA solv to the tissue and break it under ultrasonic waves until no tissue pieces are visible. Centrifuge at 10000 rpm for 5 min at 4°C. After centrifugation, aspirate the supernatant and place it on ice. The subsequent steps are consistent; 2) Add 200 μL of chloroform, tightly cap the 1.5 mL centrifuge tube, mix thoroughly for 15 s, and let it stand on ice for 10 min; 3) Centrifuge at 12000 rpm at 4°C for 15 min; 4) Carefully aspirate about 600 μL of supernatant with a pipette and transfer the aspirated supernatant to another 1.5 mL EP tube; 5) Add 1 / 2 volume of isopropanol, mix thoroughly for 15 s, and centrifuge at 12000 rpm for 1 min; 6) Place the centrifugal column HiBind® RNA Mini Column in a 2 mL collection tube, then transfer the mixed solution to the column, centrifuge at 10000 g at room temperature for 1 min, discard the flow-through and replace the collection tube; 7) Add 500 μL of RNA Wash Buffer I to the centrifugal column, centrifuge at 10000 g at room temperature for 1 min, discard the flow-through; 8) Add 500 μL of RNA Wash Buffer II to the centrifugal column, centrifuge at 10000 g at room temperature for 1 min, discard the flow-through; after washing again, discard the flow-through and replace the collection tube; 9) Add 50 μL of DEPC water to the centrifuge column and let it stand for 2 minutes. Centrifuge at 10,000 g for 1 minute at room temperature. The RNA sample is collected in the collection tube. 10) Check RNA concentration and purity: A260 / 280 ratio should be between 1.8 and 2.0. The extracted total RNA can be stored in a -80°C freezer.

[0038] Reverse transcription of RNA 1) First, remove genomic DNA contamination by taking 1000 ng of template RNA into an enzyme-free EP tube, adding Nuclease-Free Water to 12 μL, then adding 4 μL of 4 × gDNA wiper mix, gently pipetting to mix, and centrifuge briefly. 2) Incubate at 42°C for 2 minutes; 3) Add 4 μL of 5 × HiScript II qRT SuperMix II to the above reaction product, mix gently by pipetting, centrifuge briefly, incubate at 50°C for 15 min, and then at 85°C for 5 sec. Place the obtained cDNA solution on ice for subsequent use.

[0039] Quantitative Real-time PCR (qRT-PCR) Prepare a 20 μL system. The system contains 10 μL of Mix, 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L), 7 μL of deionized water, and 1 μL of 50-fold diluted cDNA. After centrifugation, follow the following protocol: 1) pre-denaturation at 95°C for 10 min, 2) denaturation at 95°C for 10 s, 3) annealing at 60°C for 30 s, and repeat 40 cycles of 2) and 3). The primer sequence table is as follows: Using 18S as the internal reference, the Ct value comparison was performed using the relative quantitative method, and the ∆Ct (Ct target gene - Ct internal reference gene) method was used for relative quantitative analysis. -∆∆Ct As the relative expression level of the target RNA.

[0040] Elisa test: Sample preparation Serum: Place the whole blood sample collected in a serum separator tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes and collect the supernatant.

[0041] 1. Take out the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature; 2. Prepare a gradient working solution of the standard: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 50 pg / mL). Then dilute to the following concentrations: 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.12 pg / mL, 1.56 pg / mL, 0.78 pg / mL, and 0 pg / mL. Serial dilution method: Take 7 EP tubes and add 500μL of universal diluent to each tube. Pipette 500μL of the 50pg / mL standard working solution into the first EP tube and mix thoroughly to make a 25pg / mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is directly used as a blank well; there is no need to pipette liquid from the penultimate tube. 3. Preparation of Biotin-antibody working solution: 15 minutes before use, centrifuge the concentrated Biotin-antibody at 1000×g for 1 minute, dilute the 100× concentrated Biotin-antibody to 1× working concentration with universal diluent, and use on the same day; 4. Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent and use on the same day. 5. Preparation of 1× washing solution: Take 10ml of 20× washing solution and add it to 190ml of distilled water. Wait until the crystals are completely dissolved before preparing.

[0042] Steps: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes; 2. Sample Addition: Dilute the test sample to a minimum of 1-fold with universal diluent before adding it to the ELISA plate. Add 50 μl of standard sample of varying concentrations to each well. Add 50 μl of universal diluent to the blank wells, followed by 50 μl of Biotin-Antibody Working Solution to each well. Cover with sealant and incubate at 37°C for 1 hour. 3. Wash the plate: Discard the liquid and add 300 μL of 1x washing solution to each well. Let it stand for 1 minute. Then, shake off the washing solution and pat dry on absorbent paper. Wash the plate 3 times. 4. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0043] 5. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 3; 6. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 20 minutes.

[0044] 7. Add stop solution: Add 50 μL of stop solution to each well, and immediately measure the OD value of each well at 450 nm wavelength.

[0045] Results are shown in Figures Figure 2 B and C, which confirmed its down-regulation in aging tissues. Considering the traditional function of ENPEP as an aminopeptidase, the levels of angiotensin II and III in the serum of young and old mice were further determined, and the results showed no significant difference between the two groups, as shown in Figure Figure 2 D, which suggests that the role of ENPEP in the aging process can be independent of the classic angiotensin metabolism function.

[0046] Example 4 Verification of the expression change of ENPEP in VSMCs in the aging process in various aging models.

[0047] Applicants gave different stimuli to the primary mouse smooth muscle, namely: hydrogen peroxide (50 μM, 24 hours), angiotensin II (5*10 7 , 5 days), D-galactose (D-gal) (20 mg / ml, 48 hours) treatment to extract protein. The cells were homogenized in RIPA buffer supplemented with protease inhibitors. The protein content was standardized using the BCA assay. The protein lysate (30 μg) in the cell sample was separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Then the PVDF membrane was incubated with a primary antibody specific for the target protein overnight at 4°C, including ENPEP, p16, p21 and β-actin. Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit) for 1 hour to analyze the protein expression level. After the treatment, RNA was extracted, reverse transcribed, and the RNA change level was detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). After the treatment, the culture medium and cells were collected, and the contents of angiotensin II and angiotensin III in the cells were detected by Elisa.

[0048] The specific method is as follows: Polyacrylamide gel electrophoresis: Applicants gave different stimuli to the primary mouse smooth muscle, namely: hydrogen peroxide (50 μM, 24 hours), angiotensin II (5*10 7Proteins were extracted after treatment with D-galactose (D-gal) (20 mg / ml, 48 hours) and RIPA buffer (RIPA, 5 days). The treated cells were sonicated in RIPA buffer supplemented with protease inhibitors. Protein content was normalized using the BCA assay. Protein lysates (30 μg) from the cell samples were separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated overnight at 4°C with primary antibodies specific for the target proteins, including ENPEP, p16, p21, and β-actin. Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit) for 1 hour. Protein expression levels were analyzed.

[0049] RNA extraction 1) Add 1 mL of RNA solution to the treated cells; 2) Add 200 µL of chloroform, tightly cap the 1.5 mL centrifuge tube, shake thoroughly for 15 seconds, and place on ice for 10 minutes; 3) Centrifuge at 12,000 rpm at 4°C for 15 min; 4) Carefully aspirate approximately 600 µL of the supernatant using a pipette and transfer the aspirated supernatant into another 1.5 mL EP tube; 5) Add 1 / 2 volume of isopropanol, vortex and mix for 15 seconds, and centrifuge at 12,000 rpm for 1 minute; 6) Place the HiBind® RNA Mini Column in a 2 mL collection tube, transfer the mixed solution to the column, and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through and replace the collection tube. 7) Add 500 μL RNA Wash Buffer I to the centrifuge column and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through. 8) Add 500 μL RNA Wash Buffer II to the centrifuge column and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through. Wash once more and discard the flow-through and replace the collection tube. 9) Add 50 μL of DEPC water to the centrifuge column and let it stand for 2 minutes. Centrifuge at 10,000 g for 1 minute at room temperature. The RNA sample is collected in the tube. 10) Check RNA concentration and purity: A260 / 280 ratio should be between 1.8 and 2.0. The extracted total RNA can be stored in a -80℃ freezer.

[0050] Reverse transcription of RNA 1) First, remove genomic DNA contamination by taking 1000 ng of template RNA into an enzyme-free EP tube, adding Nuclease-Free Water to 12 μL, then adding 4 μL of 4 × gDNA wiper mix, gently pipetting to mix, and centrifuge briefly. 2) Incubate at 42°C for 2 minutes; 3) Add 4 μL of 5 × HiScript II qRT SuperMix II to the above reaction product, gently pipette to mix, centrifuge briefly, incubate at 50°C for 15 minutes, and then incubate at 85°C for 5 seconds. Place the obtained cDNA solution on ice for subsequent use.

[0051] Quantitative Real-time PCR (qRT-PCR) Prepare a 20 μL system. The system includes 10 μL of Mix, 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L), 7 μL of deionized water, and 1 μL of cDNA at various dilutions (primer sequences as above). Mix thoroughly and centrifuge. Follow the following protocol: 1) 95°C for 10 min, 2) 95°C for 10 s, 3) 60°C for 30 s, and repeat 40 cycles of 2) and 3). Using 18S as the internal reference, the Ct value comparison was performed using the relative quantitative method, and the ∆Ct (Ct target gene - Ct internal reference gene) method was used for relative quantitative analysis. -∆∆Ct As the relative expression level of the target RNA.

[0052] SA-β-gal staining Use the β-galactosidase staining kit as follows: 1) Take out the cultured cells, discard the culture medium, and wash twice with PBS (make sure there are no dead cells under the microscope).

[0053] 2) Discard PBS and fix with 4% paraformaldehyde at room temperature for 15 minutes.

[0054] 3) Discard the fixative and wash away the fixative with PBS for 5 min x 3 times.

[0055] 4) The following steps must be protected from light. Prepare dye solution: Solution A: 10 μL, Solution B: 10 μL, Solution C: Mix well, discard PBS, and add the dye solution to the well plate until the cells are evenly covered.

[0056] 5) Incubate at 37°C, observe the staining periodically, and take photos under a bright-field microscope.

[0057] Elisa test: Sample preparation Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and collect the supernatant for testing.

[0058] Cells: Add protease inhibitors to PBS and disrupt the cells by sonication. Centrifuge the homogenate at 5000 × g for 5-10 minutes and collect the supernatant for analysis.

[0059] 1. Take out the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature.

[0060] 2. Preparation of standard working gradient solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 50 pg / mL). Then dilute to the following concentrations: 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.12 pg / mL, 1.56 pg / mL, 0.78 pg / mL, and 0 pg / mL.

[0061] Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 50 pg / mL standard working solution into the first EP tube and mix thoroughly to make a 25 pg / mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used as a blank well; there is no need to pipette liquid from the penultimate tube.

[0062] 3. Preparation of Biotin-antibody working solution: 15 minutes before use, centrifuge the concentrated Biotin-antibody at 1000×g for 1 minute. Dilute the 100× concentrated Biotin-antibody to 1× working concentration with universal diluent and use on the same day.

[0063] 4. Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent and use on the same day.

[0064] 5. Preparation of 1× washing solution: Take 10ml of 20× washing solution and add it to 190ml of distilled water. Wait until the crystals are completely dissolved before preparing.

[0065] Steps: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes.

[0066] 2. Sample Addition: Dilute the test sample to a minimum of 1-fold with universal diluent before adding to the ELISA plate. Add 50 μL of standard sample of varying concentrations to each well. Add 50 μL of universal diluent to the blank wells, followed by 50 μL of Biotin-Antibody Working Solution to each well. Cover with sealant and incubate at 37°C for 1 hour.

[0067] 3. Wash the plate: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let it stand for 1 minute. Shake off the wash buffer and pat dry on absorbent paper. Wash the plate 3 times.

[0068] 4. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0069] 5. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 3.

[0070] 6. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 20 minutes.

[0071] 7. Add stop solution: Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm.

[0072] The results showed that the expression level of ENPEP in aging VSMCs was significantly downregulated by Western blot and qRT-PCR in the VSMCs induced by hydrogen peroxide (H2O2). Figure 3 As shown in A and B, the expression of aging-related markers increased. The results of β-galactosidase staining confirmed that the aging model was successfully established. Figure 3 To exclude the influence of ENPEP's classic metabolic function, the levels of angiotensin II and III in the culture medium and VSMCs of young and aged VSMCs were further tested. The results showed that there was no significant difference between the two groups. Figure 3 To further verify this finding, the experiment was repeated in aging VSMCs models induced by angiotensin II and D-galactose, and consistent results of downregulation of ENPEP expression were observed. Figure 3 As shown in E and L. It is worth noting that in these models, the levels of angiotensin II and III in the culture medium and VSMCs were also not significantly changed, which further ruled out the possibility that ENPEP exerts its effects by regulating angiotensin II and III.

[0073] Example 5 Systematic verification of ENPEP expression changes in aging VSMCs Western blot and PCR were used to detect changes in senescence markers after transfection with si-NC and si-ENPEP, respectively. D-gal was also administered to detect the reversal of ENPEP overexpression.

[0074] The specific method is as follows: Polyacrylamide gel electrophoresis: Cell transfection in primary mouse smooth muscle: 1) Follow the instructions for Lipofectamine® 2000 Reagent. Transfect when cells in a six-well plate have grown to 70-80% confluency. 2) Add 4 μg of the calculated plasmid or 8 μl of small interfering enzyme and 8 μl of Lipo 2000 to the double-free medium, mix well, centrifuge and let stand at room temperature for 5 minutes. Add the double-free medium containing Lipo 2000 to the medium containing the plasmid, mix well, centrifuge and let stand at room temperature for 20 minutes. 3) Rinse the cells twice with PBS, add double-free culture medium, and then add the prepared mixture to the cell flask and mix well; 4) 4 hours after transfection, the minimal interference medium was replaced with complete medium and cultured for an additional 24 hours. D-gal was added to the plasmid-transfected cells for 48 hours. The treated cells were homogenized in RIPA buffer supplemented with protease inhibitors. Protein content was normalized using the BCA assay. Protein lysates (30 μg) from the cell samples were separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated overnight at 4°C with primary antibodies specific for the target proteins, including ENPEP, p16, p21, and β-actin. Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit) for 1 hour. Protein expression levels were analyzed.

[0075] RNA extraction 1) Add 1 mL of RNA solution to the treated cells.

[0076] 2) Add 200 µL of chloroform, tightly cap the 1.5 mL centrifuge tube, shake thoroughly for 15 seconds, and place on ice for 10 minutes; 3) Centrifuge at 12,000 rpm at 4°C for 15 min; 4) Carefully aspirate approximately 600 µL of the supernatant using a pipette and transfer the aspirated supernatant into another 1.5 mL EP tube; 5) Add 1 / 2 volume of isopropanol, vortex to mix for 15 seconds, and centrifuge at 12,000 rpm for 1 minute; 6) Place the HiBind® RNA Mini Column in a 2 mL collection tube, transfer the mixed solution to the column, and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through and replace the collection tube. 7) Add 500 μL RNA Wash Buffer I to the centrifuge column and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through. 8) Add 500 μL RNA Wash Buffer II to the centrifuge column and centrifuge at 10,000 g for 1 min at room temperature. Discard the flow-through. Wash once more and discard the flow-through and replace the collection tube. 9) Add 50 μL of DEPC water to the centrifuge column and let it stand for 2 minutes. Centrifuge at 10,000 g for 1 minute at room temperature. The RNA sample is collected in the tube. 10) Check RNA concentration and purity: A260 / 280 ratio should be between 1.8 and 2.0. The extracted total RNA can be stored in a -80°C freezer.

[0077] Reverse transcription of RNA 1) To remove genomic DNA contamination, transfer 1000 ng of template RNA to an enzyme-free EP tube, add Nuclease-Free Water to 12 μL, then add 4 μL of 4 × gDNA wiper mix, gently pipette to mix, and centrifuge briefly. 2) Incubate at 42°C for 2 minutes; 3) Add 4 μL of 5 × HiScript II qRT SuperMix II to the above reaction product, gently pipette to mix, centrifuge briefly, incubate at 50°C for 15 min, and then incubate at 85°C for 5 sec. Place the obtained cDNA solution on ice for subsequent use.

[0078] Quantitative Real-time PCR (qRT-PCR) Prepare a 20 μL system. This system includes 10 μL of Mix, 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L) (primer sequences are the same as above), 7 μL of deionized water, and 1 μL of cDNA at various dilutions. Mix thoroughly and centrifuge. Follow the following protocol: 1) 95°C for 10 min, 2) 95°C for 10 s, 3) 60°C for 30 s, and repeat 40 cycles of 2) and 3). Using 18S as the internal reference, the Ct value comparison was performed using the relative quantitative method, and the ∆Ct (Ct target gene - Ct internal reference gene) method was used for relative quantitative analysis. -∆∆Ct As the relative expression level of the target RNA.

[0079] Polyacrylamide gel electrophoresis: The tissue was homogenized in RIPA buffer supplemented with protease inhibitors. Protein content was normalized using the BCA assay. Protein lysates (30 μg) from the cell samples were separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated overnight at 4°C with primary antibodies specific for the target protein, including ENPEP, p16, p21, and β-actin. Subsequently, the membrane was incubated with a secondary antibody (goat anti-rabbit) for 1 hour. Protein expression levels were analyzed.

[0080] Elisa test: Sample preparation Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and remove the supernatant for detection.

[0081] Cells: Add protease inhibitors to PBS and disrupt the cells by sonication. Centrifuge the homogenate at 5000 × g for 5-10 minutes and collect the supernatant for analysis.

[0082] 1. Take out the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature.

[0083] 2. Preparation of standard working gradient solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 50 pg / mL). Then dilute to the following concentrations: 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.12 pg / mL, 1.56 pg / mL, 0.78 pg / mL, and 0 pg / mL.

[0084] Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 50 pg / mL standard working solution into the first EP tube and mix thoroughly to make a 25 pg / mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used as a blank well; there is no need to pipette liquid from the penultimate tube.

[0085] 3. Preparation of Biotin-antibody working solution: 15 minutes before use, centrifuge the concentrated Biotin-antibody at 1000×g for 1 minute. Dilute the 100× concentrated Biotin-antibody to 1× working concentration with universal diluent and use on the same day.

[0086] 4. Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent and use on the same day.

[0087] 5. Preparation of 1× washing solution: Take 10ml of 20× washing solution and add it to 190ml of distilled water. Wait until the crystals are completely dissolved before preparing.

[0088] Steps: 1. Remove the desired strips from the foil bag after equilibration at room temperature for 10 minutes.

[0089] 2. Sample Addition: Dilute the test sample to a minimum of 1-fold with universal diluent before adding to the ELISA plate. Add 50 μL of standard sample of varying concentrations to each well. Add 50 μL of universal diluent to the blank wells, followed by 50 μL of Biotin-Antibody Working Solution to each well. Cover with sealant and incubate at 37°C for 1 hour.

[0090] 3. Wash the plate: discard the liquid, add 300 μL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and wash the plate 3 times.

[0091] 4. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0092] 5. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 3.

[0093] 6. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 20 minutes.

[0094] 7. Add stop solution: Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm.

[0095] The experimental results are as follows Figure 4 As shown in Figure 3, the expression level of ENPEP is closely related to the aging process of VSMCs: when ENPEP is knocked down, the expression of aging-related marker genes such as p16 and p21 is significantly increased, as shown in Figure 3. Figure 4 As shown in A and B; overexpression of ENPEP can effectively inhibit the expression of these aging-related genes, such as Figure 4 As shown in C and D. It is worth noting that when detecting the expression levels of angiotensin II and III, it was found that neither knockdown nor overexpression of ENPEP had a significant effect on the secretion and intracellular content of angiotensin II and III, as shown in Figure 4As shown in middle E, this suggests that ENPEP may regulate VSMCs senescence through an angiotensin-independent pathway.

[0096] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications are also considered within the scope of protection of the present invention.

Claims

1. Application of preparations overexpressing ENPEP protein in anti-aging.

2. The use according to claim 1, characterized in that The ENPEP protein has the amino acid sequence shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The preparation overexpressing the ENPEP protein achieves an anti-aging effect by inhibiting the expression of p16, p21, and p53 aging-related marker genes.

4. Use of a reagent for specifically detecting the gene expression level for translating ENPEP protein in the preparation of anti-aging products.

5. The use according to claim 4, characterized in that The product includes a real-time fluorescence quantitative PCR kit.

6. The use according to claim 5, characterized in that The kit contains a primer pair as shown in SEQ ID NO.2 and SEQ ID NO.

3.

7. The use according to claim 6, characterized in that The PCR reaction system was 20 μL, and the components of the system were: PCRMix 10 μL, 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, 7 μL of deionized water, and 1 μL of cDNA; The reaction procedure was 40 cycles of pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 10 s, and annealing at 60 °C for 30 s.

8. A method for anti-aging of cells, characterized in that: The ENPEP protein is transfected into cells to construct anti-aging cells that overexpress the ENPEP protein.

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