A polypeptide upcp and its use in the preparation of a product for treating aging

Abstract

The application discloses a polypeptide UPCP and application thereof in preparation of anti-aging products. The polypeptide UPCP provided by the application has an amino acid sequence of sequence 4 in the sequence list. The polypeptide is applied in preparation of products with any one of the following functions: 1) treating children's premature aging; 2) delaying physiological aging of mammals or cells thereof; 3) improving premature aging of mammals; the improvement of premature aging of mammals is embodied in delaying premature aging of mammals; 4) improving body weight of premature aging animals; the improvement of body weight of premature aging animals is embodied in increasing body weight of premature aging animals; 5) relieving motor dysfunction of premature aging animals; 6) prolonging survival period of premature aging animals, etc. The application develops a specific peptide (UPCP) which can inhibit HGPS cell aging, improve aging characteristics of HGPS premature aging mice and prolong survival period, and provides a new strategy for treatment of HGPS.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a polypeptide UPCP and its application in the preparation of products for treating aging. Background Technology

[0002] Childhood progeria-GPS (HGPS) is a rare and severe premature aging disease with an incidence rate of approximately 1 in 4-8 million. Patients with HGPS age faster than normal, with an average lifespan of 13.4 years. HGPS is caused by a point mutation (C1824T) in the LMNA gene, affecting alternative splicing and leading to the gradual accumulation of progerin, a progerin protein with a 50-amino acid deletion at the C-terminus. Notably, progerin accumulation has also been found in the skin fibroblasts of physiologically aging individuals, and the accompanying loss of nuclear membrane proteins, nuclear membrane structure abnormalities, and mitotic abnormalities are similar to those observed in HGPS. Currently, the role and mechanism of progerin accumulation in cellular senescence remain largely unclear. Furthermore, the mechanism by which progerin accumulation causes mitotic disturbances is also poorly understood.

[0003] BUBR1, a multi-domain protein kinase, is an important component of the spindle assembly checkpoint (SAC) complex in mitosis, monitoring the correct assembly of the mitotic spindle. Studies have reported that carrying the BubR1 isotype allele mutation (BubR1...) H / H Mice exhibiting BubR1 often display aneuploid chromosomes and exhibit various age-related phenotypes. Furthermore, the levels of BubR1 in the heart, ovaries, muscles, and skin of normal mice decrease with age. Sustained high levels of BubR1 expression can prolong lifespan in mice and delay some age-related tissue degeneration, suggesting a correlation between BubR1 and cellular senescence. Childhood progeria is also considered an accelerated version of human aging, with mechanisms differing from normal aging; therefore, some indicators related to cellular senescence are not entirely applicable to childhood progeria.

[0004] Therefore, whether BUBR1 is related to childhood progeria or whether it can be used as a target to improve childhood progeria has become a research hotspot. Summary of the Invention

[0005] One object of the present invention is to provide a polypeptide.

[0006] The polypeptide (named UPCP) provided by this invention has the amino acid sequence of sequence 4 in the sequence listing.

[0007] The application of the above-mentioned polypeptide in the preparation of products having any of the functions described in 1)-12) is also within the scope of protection of this invention:

[0008] 1) Treatment of childhood progeria;

[0009] 2) Delays physiological aging in mammals or their cells;

[0010] 3) Improves premature aging in mammals; the above improvement in premature aging in mammals is specifically reflected in delaying premature aging in mammals;

[0011] 4) Improve the weight of prematurely aging animals; the above-mentioned improvement of the weight of prematurely aging animals is specifically reflected in increasing the weight of prematurely aging animals;

[0012] 5) Alleviates motor dysfunction in prematurely aging animals;

[0013] 6) Extend the lifespan of prematurely aging animals;

[0014] 7) Improve the reduction of subcutaneous fat and / or dermis in premature aging animals; specifically, improve the reduction of subcutaneous fat and / or dermis in premature aging animals by increasing the width of the epidermis or dermis or increasing the thickness of the subcutaneous fat layer.

[0015] 8) Inhibits HGPS cell senescence;

[0016] 9) Promotes HGPS cell proliferation;

[0017] 10) Inhibit aging caused by decreased BUBR1 expression;

[0018] 11) Upregulates BUBR1 expression in senescent cells;

[0019] 12) Inhibit diseases caused by the interaction between progerin and BUBR1.

[0020] Another object of the present invention is to provide a product.

[0021] The product provided by the present invention comprises the above-mentioned polypeptide; the above-mentioned polypeptide is an active ingredient.

[0022] The above products have any one of the following functions: 1)-12)

[0023] 1) Treatment of childhood progeria;

[0024] 2) Delays physiological aging in mammals or their cells;

[0025] 3) Improves premature aging in mammals; the above improvement in premature aging in mammals is specifically reflected in delaying premature aging in mammals;

[0026] 4) Improve the weight of prematurely aging animals; the above-mentioned improvement of the weight of prematurely aging animals is specifically reflected in increasing the weight of prematurely aging animals;

[0027] 5) Alleviates motor dysfunction in prematurely aging animals;

[0028] 6) Extend the lifespan of prematurely aging animals;

[0029] 7) Improve the reduction of subcutaneous fat and / or dermis in prematurely aging animals; increase the width of the epidermis or dermis or increase the thickness of the subcutaneous fat layer in the skin tissue of prematurely aging animals;

[0030] 8) Inhibits HGPS cell senescence;

[0031] 9) Promotes HGPS cell proliferation;

[0032] 10) Inhibit aging caused by decreased BUBR1 expression;

[0033] 11) Upregulates BUBR1 expression in senescent cells;

[0034] 12) Inhibit diseases caused by the interaction between progerin and BUBR1.

[0035] This invention discovers the role of BUBR1 in progerin-mediated cellular senescence and elucidates the molecular mechanism by which dysregulation and abnormal localization of BUBR1 regulate HGPS cell senescence. More importantly, this invention develops a specific peptide (UPCP) that can inhibit HGPS cell senescence, improve aging characteristics in premature HGPS mice, and prolong survival, providing a new strategy for the treatment of HGPS. Attached Figure Description

[0036] Figure 1 To anchor BUBR1 to the nuclear membrane using progerin; A: Immunofluorescence assay was performed to detect BUBR1 expression and localization in IMR90 cells overexpressing Flag-progerin, scale bars = 10 μm; B: The curve represents the fluorescence signal intensity of the dashed line in A; C: Statistical analysis of cells with BUBR1 localization on the nuclear membrane in A, ***, P < 0.001.

[0037] Figure 2To demonstrate the co-localization of BUBR1 and progerin on the nuclear membrane: A: In normal human skin fibroblast cell line CRL-1474 and HGPS patient skin fibroblast cell line HGADFN003, Western blot was used to detect the expression levels of LaminA, LaminC, and progerin, with β-actin as an internal control. B, C: In normal human skin fibroblast cell line CRL-1474 and HGPS patient skin fibroblast cell line HGADFN003, immunofluorescence was used to detect the expression and localization of BUBR1, and the cells with BUBR1 localization on the nuclear membrane were statistically analyzed. Scale bars = 50 μm, *, P < 0.05. D: In H1299 cells overexpressing mVenus-progerin, Western blot was used to detect the expression level of mVenus-progerin (mVenus tag is a variant of the GFP tag, which can be detected with GFP antibody), with β-actin as an internal control. E, F: In H1299 cells overexpressing mVenus-progerin, immunofluorescence assays were used to detect BUBR1 expression and localization, and the number of cells with BUBR1 nuclear membrane localization was statistically analyzed. Scalebars = 10 μm, ***, P < 0.001.

[0038] Figure 3 To demonstrate that progerin has a stronger affinity for BUBR1 compared to LaminA: A: In IMR90 cells stably expressing Flag-progerin, IP experiments were performed using Flag antibody, and the binding of progerin to BUBR1 was detected by Western blot. B: Purified GST-progerin was incubated with HEK-293T cell lysate, and the direct binding of progerin to BUBR1 was detected by Western blot. C: Purified GST-progerin and GST-LaminA were incubated with HEK-293T cell lysate, and the binding of progerin, LaminA, and BUBR1 was detected by Western blot. D: Flag-progerin, Flag-LaminA, and HA-BUBR1 were co-transfected into HEK-293T cells, and IP experiments were performed using Flag antibody. The binding of progerin, LaminA, and BUBR1 was detected by Western blot.

[0039] Figure 4A: Schematic diagram of the truncated domains of progerin-C (565-614aa) and BUBR1. B: Purified GST-progerin-FL, GST-progerin-N, GST-progerin-M, GST-progerin-C, and GST-LaminA-C fragments were incubated with HEK-293T cell lysates, and the binding of different truncated progerin and LaminA domains to BUBR1 was detected by Western blot. C: Flag-progerin-FL, Flag-progerin-N, Flag-progerin-M, Flag-progerin-C, and Flag-LaminA-C domains were transfected into HEK-293T cells, and IP assays were performed using Flag antibody. The binding of different truncated progerin and LaminA domains to BUBR1 was detected by Western blot.

[0040] Figure 5 To block the interaction between progerin and BUBR1 using progerin-C: A: HEK-293T cells were transfected with Flag-progerin and Flag-progerin-C, respectively. Subcellular localization of progerin and progerin-C was detected by immunofluorescence assay (scale bars = 100 μm). B: HEK-293T cells were co-transfected with HA-progerin (10 μg) and different doses of Flag-progerin-C (0 μg, 5 μg, 10 μg, 15 μg). Intracellular immunoassay (IP) was performed using Flag and HA antibodies, respectively. Western blot analysis was used to detect the binding of progerin and progerin-C to BUBR1.

[0041] Figure 6 A: Schematic diagram of different progerin C-terminal truncation to block the interaction between progerin and BUBR1. B: HA-progerin and different Flag-progerin C-terminal fragments were co-transfected in HEK-293T cells. IP experiments were performed using HA antibody, and Western blot was used to detect the effect of different progerin C-terminal peptide fragments on the binding of progerin and BUBR1.

[0042] Figure 7To block the interaction between progerin and BUBR1 using UPCP; A: Amino acid sequences of progerin-C36 and UPCP peptides. B: Immunofluorescence assay to detect UPCP uptake in HGADFN003 cells at different time points, scale bars = 50 μm. C: HEK-293T cells were transfected with HA-progerin, and treated with different concentrations of UPCP (0 μM, 6 μM, 12 μM, 18 μM) for 48 h. IP assay was performed using HA antibody, and Western blot was used to detect the effect of UPCP on the binding of progerin and BUBR1.

[0043] Figure 8 To investigate the effects of UPCP on BUBR1 expression: A: Western blot analysis of the effect of different progerin-C fragments on BUBR1 expression in the HGADFN003 skin fibroblast cell line from HGPS patients. B: Western blot analysis of the effect of different concentrations of UPCP on BUBR1 expression in the HGADFN003 skin fibroblast cell line from HGPS patients. C: Immunofluorescence assay of the effect of UPCP on BUBR1 expression in the HGADFN003 skin fibroblast cell line from HGPS patients (scale bars = 50 μm). D: Western blot analysis of the effect of UPCP on BUBR1 expression in the IMR90-progerin cell model.

[0044] Figure 9To investigate the antagonism of HGPS cell senescence by UPCP: A: In the HGPS patient skin fibroblast cell line HGADFN003, Western blot was used to detect the effect of UPCP (6 μM) on the expression levels of cellular senescence markers HP1γ, H3K9me3, and CyclinA2. B: In the HGPS patient skin fibroblast cell line HGADFN003, SA-β-gal staining was used to detect the effect of UPCP on HGADFN003 cell senescence and the results were statistically analyzed. **, P<0.01. C, D: In the HGPS patient skin fibroblast cell line HGADFN003, immunofluorescence assay was used to detect the effect of UPCP on the expression of the cell proliferation marker Ki67 and the results were statistically analyzed. Scale bars = 20 μm, *, P<0.05. E: In the HGPS patient skin fibroblast cell line HGADFN167, SA-β-gal staining was used to detect the effect of UPCP on HGADFN167 cell senescence and the results were statistically analyzed. **, P<0.01. F, G: In the HGADFN167 skin fibroblast cell line of HGPS patients, the effect of UPCP on the expression of cell proliferation marker Ki67 was detected by immunofluorescence assay and statistically analyzed. Scale bars = 20 μm, **, P < 0.01.

[0045] Figure 10 To investigate how UPCP antagonizes HGPS cell senescence via BUBR1: A: In the HGPS patient skin fibroblast cell line HGADFN003 treated with UPCP (6 μM), endogenous BUBR1 was knocked down. Western blot was used to detect the expression levels of BUBR1, LaminB1, CyclinA2, and H3K9me3. B, C: In the HGPS patient skin fibroblast cell line HGADFN003 treated with UPCP, endogenous BUBR1 was knocked down. SA-β-gal staining was used to detect and statistically analyze the senescence status of HGADFN003 cells. Scale bars = 100 nm**, P < 0.01, ***, P < 0.001. D, E: In the HGPS patient skin fibroblast cell line HGADFN003 treated with UPCP, endogenous BUBR1 was knocked down. Immunofluorescence assay was used to detect and statistically analyze Ki67 expression. Scale bars = 50 μm, *, P < 0.05, **, P < 0.01. F: In the HGADFN003 skin fibroblast cell line of HGPS patients treated with UPCP, endogenous BUBR1 was knocked down, and chromosome karyotype analysis was performed to detect aneuploid cells and the results were statistically analyzed. **, P<0.01.

[0046] Figure 11To antagonize physiological cellular senescence with UPCP: A: In skin fibroblasts of a 92-year-old patient, UPCP (6 μM) was treated for 72 h, and the expression levels of BUBR1, Cyclin A2, and H3K9me3 were detected by Western blot. B: In skin fibroblasts of a 92-year-old patient, UPCP was treated for 72 h, and the physiological cellular senescence was detected and statistically analyzed by SA-β-gal staining. *, P<0.05. C, D: In skin fibroblasts of a 92-year-old patient, UPCP was treated for 72 h, and the expression of Ki67 was detected and statistically analyzed by immunofluorescence assay. Scale bars = 50 μm, **, P<0.01.

[0047] Figure 12 UPCP showed no significant toxicity to cells. A: In IMR90 cells, Western blot was used to detect the effect of UPCP on the expression of the cell proliferation marker Cyclin A2. B, C: In IMR90 cells, immunofluorescence assays were used to detect and statistically analyze Ki67 expression. Scale bars = 20 μm, ns, no significant difference was observed. D: IMR90 cells were seeded in 96-well plates and cultured for 24 h. Different concentrations of UPCP (0 μM, 10 μM, 20 μM, 30 μM) were added. OD values ​​were measured at 0 h, 24 h, 48 h, and 72 h, ns, no significant difference was observed. E: IMR90 cells were seeded in 96-well plates, and different concentrations of UPCP (0 μM, 10 μM, 20 μM, 30 μM) were added the next day. OD values ​​were measured after 48 h of culture. Cell viability was calculated as OD value of the UPCP treatment group / OD value of the control group, ns, no significant difference was observed.

[0048] Figure 13 To improve body weight in premature aging mice using UPCP; A: Female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Representative morphological images of mice after 12 weeks. B: Female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Changes in body weight in mice, n = 6, 9, 9. C: Female Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Weight changes at 4 and 12 weeks, n = 6, 9, 9, ns, no significant difference, *, P < 0.05. D: Male Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609GRepresentative morphological images of mice after 12 weeks. E: Male Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Changes in mouse body weight, n = 11, 8, 6. F: Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Weight changes at 4 and 12 weeks, *, P<0.05, **, P<0.01.

[0049] Figure 14 To prolong the survival of premature aging mice using UPCP; A: Female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Kaplan-Meier survival curves for mice, n = 9, 9, 9, ****, P < 0.0001. B: Male Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Kaplan-Meier survival curves for mice, n = 9, 8, 6, **, P < 0.01.

[0050] Figure 15 To alleviate motor dysfunction in premature aging mice using UPCP; A: Female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Visual images of mouse open field movement trajectories and statistics of total movement distances after 12 weeks, n = 6, 6, 6, ***, P < 0.001, ****, P < 0.0001. B: Male Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Visual images of mouse movement trajectories and statistics of total movement distances after 12 weeks in the open field experiment, n = 6, 6, 6, ***, P < 0.001, ****, P < 0.0001.

[0051] Figure 16 To increase subcutaneous fat in premature aging mice using UPCP; A: Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Representative images of mouse skin stained with hematoxylin and eosin (HE) at 13 weeks post-mortem. Dashed squares and single arrows represent magnified areas of the epidermis, while double arrows represent subcutaneous fat layers. Scale bars = 250 μm. B: For Lmna + / + Lmna G609G / G609G and UPCP treatment for LmnaG609G / G609G Statistical analysis was performed on the fat layer of mouse skin tissue, n=3, *, P<0.05, ****, P<0.0001. Detailed Implementation

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0053] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] The relevant information regarding the antibodies used in the following examples is shown in Table 1 below:

[0055] Table 1 is a summary table of antibodies.

[0056]

[0057] The following cell lines were used in the examples: human embryonic kidney cells HEK293T, human skin fibroblasts CRL-1474, and human embryonic lung fibroblasts IMR90. All cell lines were purchased from the American Type Culture Collection (ATCC). HGADFN003 and HGADFN167 skin fibroblasts from HGPS (Hypergeria GPS) patients were obtained from The Progeria Research Foundation (PRF). AG09602 skin fibroblasts from a 92-year-old patient were obtained from the Aging Respository of the Coriell Cell Respository (CCR) at the Carrier Institute for Medical Research.

[0058] The mice used in the experiment were all fed according to a 12-hour diurnal rhythm, and the experimental procedures complied with the animal use guidelines of Northeast Normal University.

[0059] The following examples use live animals of the ICR strain, Lmna. G609G / G609G The premature aging mouse model was prepared according to the following method:

[0060] Lmna + / G609GMale and female mice were hybridized. Genotyping of the mice was performed using a Mouse Direct PCR Kit (For Genotyping) (Bimake 820049) for PCR fragment amplification. The primer sequences were: mLMNA_fwd:CGAAGGCTTCCTGGCTATTT; mLMNA_rev:TGCTGTAGGGCAGAGATGA. The target fragment was then recovered and sequenced by Kumei Pharmaceuticals to identify the mouse genotype. Mice with the genotype G609G / G609G were selected as Lmna mice. G609G / G609G A mouse model of premature aging.

[0061] The above Lmna + / G609G The mice were obtained by replacing the Lmna gene (Gene ID: 169052022-2-6) in the genome of ICR wild-type mice with an allele carrying the c.1827C>T; p.G609G mutation (the C at position 1827 of the sequence shown in genbank Gene ID: 16905, 2022-2-6 is mutated to T). The ICR wild-type mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0062] The Realtime PCR primer sequences used in the following examples are shown in Table 2 below:

[0063] Table 2 shows the primer sequences.

[0064]

[0065] The experimental methods in the following examples are as follows:

[0066] I. Protein Purification Experiment

[0067] For conventional methods, please refer to the following reference (Liu, J., Feng, J., Li, L., Lin, L. & Lu, J. Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer. EMBO Reports 21, e48597 (2019).).

[0068] II. GST pull-down experiment

[0069] For conventional methods, please refer to the following reference (Liu, J., Feng, J., Li, L., Lin, L. & Lu, J. Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer. EMBO Reports 21, e48597 (2019).).

[0070] III. Co-immunoprecipitation assay (Co-IP)

[0071] (1) Collect cells.

[0072] (2) Discard the supernatant, add 1 mL of Co-IP buffer to the cell pellet, lyse on ice for 30 min, and vortex for 1 min every 2 min.

[0073] (3) After cell lysis, centrifuge at 4°C for 10 min at 12,000 rpm.

[0074] (4) Transfer 1 mL of supernatant to a new 1.5 mL centrifuge tube, take 100 μL of it as input sample, add an appropriate amount of loading buffer and mix well, and boil in a 100 °C metal bath for 8-10 min. Then store the input protein sample at -20 °C for later use.

[0075] (5) Add 5 μg of antibody to the remaining sample, seal with sealing film and incubate overnight at 4°C on a vertical mixer.

[0076] (6) On the second day, add 30 μL of pre-equilibrated protein A / G magnetic beads (Millipore LSKMAGAG02) to the sample and incubate at 4°C for 3 h or at room temperature for 1.5 h on a vertical mixer.

[0077] (7) Use a magnetic rack to remove the supernatant, wash the magnetic beads with PBS for 8 min / time, 3 times.

[0078] (8) Add 50 μL Loading Buffer, mix thoroughly, boil in a 100°C metal bath for 8 min, transfer the supernatant to a new centrifuge tube using a magnetic rack, and store at -20°C for later use.

[0079] The reagent formulations used in this experiment are shown in Table 3 below:

[0080] Table 3 shows the Co-IP Buffer (100 mL).

[0081]

[0082] Dissolve in dd H2O and bring the volume to 100 mL. Store at 4°C for later use. When using Co-IP Buffer, add the protease inhibitors (A, P, L, DTT, and PMSF) according to the specified ratio.

[0083] IV. Immunofluorescence Experiment

[0084] For routine experiments, please refer to the following reference (Lin, C., et al. Arginine hypomethylation-mediated proteasomal degradation of histone 737H4—an early biomarker of cellular senescence. Cell Death & Differentiation 27, 2697-2709 738 (2020)).

[0085] V. Chromosome Karyotype Analysis Experiment

[0086] 1. Seed the target cells in 6cm plates (for example, the seeding density for IMR90 cells is 1.5 × 10⁻⁶). 6 indivual).

[0087] 2. After 24 hours, the cells were in good condition with high density and good refractive properties. Colchicine was added to the cells at a final concentration of 0.2 μg / ml, and the culture was terminated after 2 hours of treatment.

[0088] 3. Digest the cells from step 2 above, collect the target cells, centrifuge them at 1000 rpm for 5 minutes, and then discard the supernatant.

[0089] 4. Add 1 mL of preheated hypotonic solution (0.075 mol / L) at 37°C to the centrifuge tube for resuspending, mix well by blowing, and incubate in a 37°C water bath for 20 min.

[0090] 5. Centrifuge and discard the supernatant. Add 0.5-1 mL of fixative along the wall of the dorf tube, gently resuspend the cells, incubate at room temperature for 10 min, centrifuge and discard the supernatant.

[0091] 6. Add 1 mL of fixative to the centrifuge tube, let stand at room temperature for 30 min, centrifuge and discard the supernatant.

[0092] 7. Repeat the above 6 steps once.

[0093] 8. Add 0.5 mL of fixative to the centrifuge tube to resuspend the cells.

[0094] 9. Use a pipette to draw an appropriate amount of cell suspension, drop the cell suspension onto a pre-cooled glass slide from a height, pass the cell-free side of the glass slide over the flame of an alcohol lamp several times, and dry it in an oven.

[0095] 10. DAPI staining solution can be used to stain cell nuclei spread on a glass slide, the slide can be mounted, and images can be taken under a microscope.

[0096] 11. The reagent formulas used in this experiment are as follows:

[0097] (1) Hypotonic solution (200mL)

[0098] Table 4 shows the formulations for hypotonic solutions.

[0099]

[0100] (2) Fixative (4 mL, prepare fresh before use)

[0101] Table 5 shows the formulations of the fixative.

[0102]

[0103] VI. Behavioral Analysis of Mice

[0104] This experiment will use the open field test to assess the motor ability of mice. The specific steps are as follows:

[0105] 1. Place the mice in the experimental environment one day in advance to allow them to become familiar with the environment (the experiment is conducted in a quiet environment).

[0106] 2. Place the mouse in the center of the bottom of the box, and the camera will capture images and stop recording after a certain time. (After each mouse finishes the experiment, clean the inner wall and bottom of the box in time to avoid affecting the next mouse.)

[0107] 3. Different parameters are detected using computer software, and the corresponding data is obtained and analyzed.

[0108] Example 1: Discovery and preparation of a novel peptide UPCP

[0109] I. Discovery of the segment blocking progerin binding to BUBR1

[0110] 1. BUBR1 is abnormally localized in HGPS cells and is anchored to the nuclear membrane by progerin.

[0111] IMR90 cells expressing progerin: The progerin gene LMNA (GeneID:4000, updated on 2021-12-5, lamin isoform A-delta50) was inserted between the BglII and EcoR1 sites of the pWZL-mVenus vector (this plasmid was obtained by replacing the mVenus base sequence (sequence 2) containing the multiple cloning site sequence between the BamHI and SalI restriction sites of the vector pWZL hygro (Addgene#18750)). The recombinant vector was then packaged with virus and infected into IMR90 cells (ATCC, CCL-186) to obtain IMR90 cells expressing mVenus-progerin.

[0112] Immunofluorescence assays were performed on IMR90 cells overexpressing mVenus-progerin (antibody: BUBR1-specific antibody, Abcam ab54894). The results confirmed that in the control group (IMR90 cells transfected with pWZL-mVenus vector, shown in the image as mVenus), BUBR1 was subcellularly distributed in the cytoplasm, while in interphase cells expressing progerin (IMR90 cells overexpressing mVenus-progerin, shown in the image as mVenus-progerin), BUBR1 was anchored to the nuclear membrane and co-localized with progerin. Figure 1 AC).

[0113] The same phenomenon was also observed in CRL-1474 and HGADFN003 cells, as well as in H1299 cells that underwent exogenous overexpression of mVenus-progerin using the same method. Figure 2 AF).

[0114] 2. BUBR1 binds to progerin and specifically binds to the C-terminal domain of progerin.

[0115] 1) BUBR1 binds to progerin

[0116] It has been shown that BUBR1 and progerin are colocalized on the nuclear membrane. So, are they in the same complex?

[0117] The progerin gene LMNA (GeneID: 4000, updated 2021-12-5, lamin isoform A-delta50) was inserted between the BamHI and NotI sites of the pGEX-6P1 vector (GE Healthcare, 28-9546-48) to obtain a recombinant vector expressing GST-progerin; this vector was then transformed into BL21 Escherichia coli competent cells to obtain recombinant bacteria expressing GST-progerin; GST-progerin was purified from this recombinant bacteria.

[0118] 5 μg of purified GST-progerin (with a GST tag attached to the N-terminus of the progerin) was mixed with 500 ng / ml of HEK293T cell lysis buffer (CoIP buffer) on ice. HEK293T cells were lysed on ice, as detailed in the experimental methods above: Immunoprecipitation experiment (1)-(4)). The mixture was placed in a centrifuge tube and mixed on a vertical mixer. The mixture was incubated overnight at 4°C. Western blot was used to detect whether progerin and BUBR1 directly bound. The results showed that progerin and BUBR1 could directly bind to each other. Figure 3 A).

[0119] The progerin gene LMNA (GeneID: 4000, updated on 2021-12-5, lamin isoform A-delta50) was inserted into the pCDH-CMV-3xFlag vector (this plasmid vector is obtained by replacing the 3xFlag base sequence (sequence 3) containing the multiple cloning site sequence with the XbaI and NotI restriction sites of the vector pCDH-CMV-MCS-EF1-puro CD510B-1 (HH-LV-002)) between the BamHI and NotI sites, resulting in a recombinant vector expressing Flag-progerin;

[0120] In IMR90 cells stably expressing Flag-progerin (after transfection of IMR90 cells with a recombinant vector expressing Flag-progerin), IP assays were performed using Flag antibody, and Western blot analysis was used to detect the binding of progerin to BUBR1, confirming the interaction between progerin and BUBR1. Figure 3 B).

[0121] 2) BUBR1 binds to LaminA

[0122] Progerin, a dominant-negative effector protein of LaminA, influences various intracellular physiological events. Experiments have shown that progerin and BUBR1 can directly bind to each other. Does LaminA interact with BUBR1?

[0123] The LaminA gene LMNA (GeneID:4000, 2021-12-5) was inserted between the BamHI and NotI sites of the pGEX-6P1 vector (GEHealthcare, 28-9546-48) to obtain a recombinant vector expressing GST-LaminA; this vector was then transformed into BL21 Escherichia coli competent cells to obtain recombinant bacteria expressing GST-LaminA; GST-LaminA was purified from this recombinant bacteria.

[0124] GST-progerin and GST-LaminA were purified in vitro and mixed with HEK293T cell lysis buffer (CoIP buffer) on ice. HEK293T cells were lysed (see the experimental methods above: Immunoprecipitation experiment (1)-(4)) in centrifuge tubes placed on a vertical mixer and incubated overnight at 4°C. Western blot analysis was performed (antibody was BUBR1, ab209998). The results showed that LaminA also interacted with BUBR1, but its affinity was significantly weaker than that of progerin. Figure 3 C).

[0125] In HEK293T cells, recombinant vectors expressing Flag-progerin, Flag-LaminA (LaminA gene inserted between the BamHI and NotI sites of the pCDH-CMV-3xFlag vector), and HA-BUBR1 (BUBR1 gene inserted between the BamHI and NotI sites of the pcDNA3.1(+)(Invitrogen, V790-20) vector) were co-transfected. IP experiments were performed using Flag antibody, and the binding of progerin, LaminA, and BUBR1 (antibodies Flag and BUBR1) was detected by Western blot. The same results were confirmed in endogenous CoIP experiments, indicating that the interaction between progerin and BUBR1 was significantly stronger than the interaction between LaminA and progerin. Figure 3 D).

[0126] To further explain this difference in affinity, LaminA (LMNAGeneID:4000, updated on December 5, 2021) and progerin were truncated to obtain progerin-FL (progerin amino acid sequence (Sequence 1) 1-614 aa), progerin-N (Sequence 1 1-300 aa), progerin-M (Sequence 1 301-564 aa), progerin-C (Sequence 1 565-614 aa), and LaminA-C (GeneID:4000, updated on December 5, 2021, amino acid sequence 565-664 aa). Figure 4 A).

[0127] The coding genes of the above truncated somatic proteins were respectively linked between the BamHI and NotI sites of the pGEX-6P1 vector to obtain the related vectors with GST tags - truncated somatic proteins.

[0128] Alternatively, the coding genes of these proteins can be linked between the BamHI and NotI sites of the pCDH-CMV-3xFlag vector to obtain related vectors with Flag tag-truncated proteins.

[0129] The GST-tagged vectors were then transformed into BL21 Escherichia coli competent cells to obtain recombinant bacteria. The recombinant bacteria were then purified according to the method described above to obtain progerin-FL, progerin-N, progerin-M, progerin-C, and LaminA-C segments with GST tags. All of these tags are attached to the N-terminus of the protein.

[0130] Purified GST-progerin-FL, GST-progerin-N, GST-progerin-M, GST-progerin-C, and GST-LaminA-C fragments were mixed with HEK293T cell lysate in centrifuge tubes and incubated overnight at 4°C. Western blot analysis was used to detect the binding of different truncated progerin and LaminA fragments (antibody: BUBR1) to BUBR1. The common region M (301-564 aa) of LaminA and progerin bound to BUBR1. The progerin-C fragment (565-614 aa) specifically bound to BUBR1, while the LaminA-C fragment (565-664 aa) did not bind to BUBR1. Figure 4 B).

[0131] HEK293T cells were transfected with vectors containing Flag-progerin-FL, Flag-progerin-N, Flag-progerin-M, Flag-progerin-C, and Flag-LaminA-C, respectively. In vitro immunoassay (IP) was performed using Flag antibodies, and Western blot was used to detect the binding of different truncated progerin and LaminA to BUBR1. The same results were confirmed by endogenous CoIP assays. Figure 4 C).

[0132] In summary, the results indicate that BUBR1 can specifically bind to the C-terminal domain of progerin, resulting in a significantly stronger affinity between progerin and BUBR1 than between LaminA and BUBR1.

[0133] 3. Progerin-C interferes with the interaction between BUBR1 and progerin.

[0134] It has been shown that progerin binding to BUBR1 inhibits BUBR1 function, so interfering with progerin binding to BUBR1 is likely a strategy to restore BUBR1 function.

[0135] It has been demonstrated that the progerin-C segment can specifically bind to BUBR1, therefore it is speculated that the progerin-C segment may compete with progerin for binding to BUBR1.

[0136] To verify this hypothesis, HEK293T cells were transfected with the Flag-progerin vector and Flag-progerin-C, as follows:

[0137] (1) Seed HEK-293T cells in 10cm plates. The cell density should reach 40-50% on the second day. (2) On the second day, replace the culture medium of HEK-293T cells with 5mL of serum-free and antibiotic-free DMEM medium. (3) Incubate the recombinant vector expressing Flag-progerin or the recombinant vector expressing Flag-progerin-C and the transfection reagent PEI in 1.5mL dorf tubes containing 500μL of serum-free and antibiotic-free DMEM medium, respectively. The ratio of plasmid to PEI (mass:volume) is 3:1. Mix thoroughly and incubate for 15-30min. (4) After incubation, add the above liquid dropwise to the cells. Mix well and place in a 37°C constant temperature incubator to obtain HEK293T cells transfected with Flag-progerin and HEK293T cells transfected with Flag-progerin-C.

[0138] Immunofluorescence was used to detect the subcellular localization of progerin and progerin-C in HEK293T cells transfected with Flag-progerin and Flag-progerin-C. The results showed that progerin-C was distributed throughout the cell, while progerin was localized on the nuclear membrane. Figure 5 A).

[0139] Next, in order to further explore the effect of progerin-C on the interaction between progerin and BUBR1,

[0140] 10 μg of a recombinant vector expressing HA-progerin (constructed using the same method as above) and different doses (0 μg, 5 μg, 10 μg, and 15 μg) of a recombinant vector expressing Flag-progerin-C were co-transfected into HEK293T cells. In vitro immunoassay (IP) experiments were performed using Flag and HA antibodies, respectively. Western blot analysis was used to detect the binding of progerin and progerin-C to BUBR1. The results showed that as progerin-C gradually increased, the binding between progerin and BUBR1 gradually weakened, while the binding between progerin-C and BUBR1 gradually strengthened. Figure 5 B).

[0141] The above experimental results indicate that progerin-C can block the interaction between progerin and BUBR1.

[0142] II. Acquisition of UPCP and its Interference: BUBR1 and Progerin Interaction

[0143] 1. Obtaining UPCP

[0144] It has been confirmed that the C-terminus of progerin (sequence 1, amino acids 565-614aa) can effectively block the binding of progerin to BUBR1. To further confirm the key functional regions in the 50 amino acids at the C-terminus of progerin, the C-terminus of progerin was truncated to obtain progerin-C50 (sequence 1, amino acids 565-614aa), progerin-C45 (sequence 1, amino acids 570-614aa), progerin-C36 (sequence 1, amino acids 579-614aa), and progerin-C27 (sequence 1, amino acids 588-614aa).

[0145] The coding genes for the truncated somatic protein were then ligated between the BamHI and NotI sites of the pCDH-CMV-3xFlag vector to obtain a vector containing the Flag tag-truncated somatic protein. Figure 6 A).

[0146] Recombinant vectors expressing HA-progerin and recombinant vectors expressing different Flag-progerin-C truncated proteins were co-transfected into HEK293T cells. IP assays were performed using HA antibody, and Western blot was used to detect the effects of different progerin-C terminal peptides on the binding of progerin and BUBR1. The results showed that different progerin-C fragments could inhibit the binding of progerin and BUBR1 to some extent, with progerin-C36 being particularly effective. Figure 6 B).

[0147] Next, the focus shifted to progerin-C36, and a progerin-C peptide with the corresponding number of amino acids was synthesized. To increase the transmembrane capacity and visualization of the peptide, an 11-amino acid HIV-TAT sequence was added to the N-terminus of the progerin-C36 amino acid sequence, and a FITC modification group was added to the N-terminus. This peptide was named the Unique Progerin C-terminal Peptide (UPCP). Figure 7 A) The amino acid sequence of this peptide is sequence 4.

[0148] UPCP is composed of 47 L-type amino acids, including the transmembrane peptide HIV-TAT, and has the molecular formula C. 211 H 360 N 80 O 66 S4.

[0149] The amino acid sequence of UPCP is as follows: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQSPQNCSIM (Sequence 4).

[0150] The aforementioned UPCP can be prepared through artificial synthesis or prokaryotic expression.

[0151] 2. UPCP inhibits the interaction between progerin and BUBR1.

[0152] 1) Cellular uptake of UPCP

[0153] UPCP modified with an N-terminus linked to a FITC group was added to DMEM complete medium (final concentration 6 μM), and HGADFN003 cells were cultured in this medium. Immunofluorescence assays were performed to detect the uptake of UPCP by HGADFN003 cells at different time points (since FITC is a green fluorescent protein, it exhibits green fluorescence under UV excitation; therefore, no antibody was used, only DAPI staining and fluorescence signal collection under a microscope). The results showed that UPCP could be taken up by cells after 2 hours of culture and remained stable in cells for at least 48 hours. Figure 7 B).

[0154] 2) Effects of UPCP on the interaction between progerin and BUBR1

[0155] HEK293T cells were transfected with a recombinant vector expressing HA-progerin to obtain HEK293T cells transfected with HA-progerin.

[0156] HEK293T cells transfected with HA-progerin were cultured in medium containing different concentrations of UPCP (0 μM, 6 μM, 12 μM, 18 μM) for 48 h. In vitro immunoassay (IP) was performed using HA antibody, and Western blot was used to detect the effect of UPCP on the binding of progerin and BUBR1. CoIP experiments showed that the binding ability of progerin to BUBR1 gradually weakened with increasing UPCP concentration, indicating that UPCP can inhibit the interaction between progerin and BUBR1. Figure 7 C).

[0157] Based on the above experimental results, UPCP can block the interaction between progerin and BUBR1.

[0158] Recombinant vectors expressing different flag tag progerin-C segments were transfected into the HGADFN003 skin fibroblast line from HGPS patients. Western blot analysis was used to examine the effect of different progerin-C segments on BUBR1 expression. The results showed that different progerin-C segments could upregulate BUBR1 expression to some extent, with progerin-C36 being particularly effective. Figure 8 A).

[0159] The HGADFN003 skin fibroblast cell line from HGPS patients was cultured for 72 h in medium containing different concentrations of UPCP (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM). Western blot analysis (antibody: BUBR1) was used to detect the effect of different concentrations of UPCP on BUBR1 expression. The results showed that BUBR1 expression in HGPS patient skin fibroblasts increased with increasing UPCP concentration. Figure 8 B).

[0160] The HGADFN003 skin fibroblast line from HGPS patients was cultured for 72 hours in a medium containing 6 μM of UPCP modified with an N-terminus linked to a FITC group. Immunofluorescence assays (antibody: BUBR1) were used to detect the effect of UPCP on BUBR1 expression. The immunofluorescence results also confirmed that UPCP could upregulate BUBR1 expression. Figure 8 C).

[0161] The IMR90-progerin cell model (i.e., IMR90 cells expressing progerin) was cultured in UPCP medium with 6 μM concentration for 72 h. Western blot analysis was used to detect the effect of UPCP (antibody: BUBR1) on BUBR1 expression. UPCP was found to upregulate BUBR1 expression in the IMR90-progerin cell model. Figure 8 D).

[0162] The experimental data above show that UPCP can upregulate BUBR1 expression.

[0163] Example 2: UPCP inhibits aging

[0164] I. UPCP inhibits HGPS cell senescence

[0165] 1. UPCP antagonizes HGPS cell senescence

[0166] It has been confirmed that UPCP can thaw the interaction between progerin and BUBR1 and upregulate the abundance of BUBR1. The next step is to further investigate the effect of UPCP on cell senescence.

[0167] 1) UPCP processing

[0168] HGADFN003 skin fibroblast cell line from HGPS patients was cultured in medium containing 6 μM UPCP for 72 h to obtain HGADFN003 cells that took up UPCP.

[0169] 2) Western blot detection

[0170] HGADFN003 cells that had taken up UPCP after being cultured for 72 hours were used in Western blot to detect the effects of UPCP (HP1γ antibody, H3K9me3 antibody, and Cyclin A2) on the expression levels of cellular senescence markers HP1γ, H3K9me3, and Cyclin A2. The results are as follows: Figure 9 As shown in Figure A, the expression of HP1, H3K9me3, and Cyclin A2 was low in the HGPS aging model. However, the addition of UPCP to skin fibroblasts from HGPS patients upregulated the expression levels of HP1, H3K9me3, and Cyclin A2, thereby inhibiting cellular senescence. Figure 9 A).

[0171] 3) SA-β-gal staining detection

[0172] HGADFN003 cells that have taken up UPCP and were cultured for 72 hours were stained with SA-β-gal. The more SA-β-gal positive cells there were, the stronger the degree of cell senescence.

[0173] The results are as follows Figure 9 As shown in Figure B, it can be seen that, compared with not adding UPCP (MOCK), adding UPCP can inhibit the senescence of skin fibroblasts in HGPS patients. Figure 9 B).

[0174] 4) Immunofluorescence detection of cell proliferation marker Ki67

[0175] The effect of UPCP uptake on the expression of the cell proliferation marker Ki67 in HGADFN003 cells obtained by culturing for 72 h as described above was detected by immunofluorescence assay. Ki67 was used as the antibody.

[0176] The results are as follows Figure 9 C and Figure 9 As shown in Figure D, it can be seen that, compared with no UPCP (MOCK), adding UPCP can promote the expression of marker Ki67 in HGADFN003 cells. Figure 9 C), that is, UPCP can increase the proportion of Ki67 positive cells in HGADFN003 cells ( Figure 9 D). Simultaneously, the above experiment was performed using another HGPS cell line, HGADFN167 cells, and the results were consistent with those obtained in HGADFN003 cells. Figure 9 E-9G).

[0177] In summary, UPCP can delay HGPS cell senescence or promote HGPS cell proliferation, thereby inhibiting HGPS cell senescence.

[0178] 2. UPCP inhibits senescence in HGPS cells by regulating BUBR1.

[0179] To further evaluate the direct role of UPCP in regulating BUBR1 in HGPS cell senescence, BUBR1 expression was interfered in UPCP-treated HGADFN003 skin fibroblasts from HGPS patients, thereby detecting the cellular senescence phenotype.

[0180] 1) UPCP processing

[0181] HGADFN003 cells were cultured in a medium containing 6 μM UPCP for 72 h to obtain HGADFN003 cells that took up UPCP.

[0182] 2) Preparation of shBUBR1 virus for interfering with BUBR1 expression

[0183] The pLKO.1-BUBR1#3 lentiviral interference plasmid is a plasmid in which the shBUBR1#3 interference fragment is ligated between the AgeI and EcoRI sites of the pLKO.1 vector (addgene#10878).

[0184] The pLKO.1-control lentiviral interference plasmid is formed by ligating the control interference fragment between the AgeI and EcoRI sites of the pLKO.1 vector (addgene#10878).

[0185] The above-mentioned shBUBR1#3 interference fragments were prepared by annealing shBUBR1#3-sense and shBUBR1#3-antisense from Table 6.

[0186] The aforementioned control interference fragments were prepared by annealing the shCtrl-sense and shCtrl-antisense fragments from Table 6.

[0187] The aforementioned interference plasmids were then transformed into HEK-293T cells, and packaged to obtain viruses infecting shBUBR1 and control viruses.

[0188] Table 6 shows the interference primer sequences:

[0189]

[0190] The aforementioned interference plasmids were transfected into HEK-293T cells to prepare viruses, and the viruses were then used to infect HGADFN003 cells using standard methods. (Reference: Lin, C., et al. Arginine hypomethylation mediated proteasomal degradation of histone H4 an early biomarker of cellular senescence. Cell Death & Differentiation Differentiation 27, 2697-2709 (2020)).

[0191] 3) Verification

[0192] Proteins were extracted from HGADFN003 cells (UPCP+shBUBR1#3) with knocked-down endogenous BUBR1 who took up UPCP to verify infection efficiency. HGADFN003 control cells (UPCP+shCtrl) with UPCP took up UPCP served as controls.

[0193] Western blot analysis was performed on BUBR1 gene expression in HGADFN003 cells that had taken up UPCP and had endogenous BUBR1 knocked down. The results are as follows: Figure 10 As shown in Figure A, compared with HGADFN003 control cells that take up UPCP (UPCP+shCtrl), BUBR1 expression was reduced in HGADFN003 cells that take up UPCP and have their endogenous BUBR1 knocked down (UPCP+shBUBR1#3).

[0194] The above results indicate that HGADFN003 cells with knockdown of endogenous BUBR1 were successfully constructed.

[0195] 4) Western blot method for detecting aging-related markers

[0196] Western blot analysis was used to detect the expression levels of LaminB1, CyclinA2, and H3K9me3 in HGADFN003 cells that took up UPCP and had their endogenous BUBR1 knocked down, and in HGADFN003 control cells that took up UPCP.

[0197] The results are as follows Figure 10As shown in Figure A, compared with HGADFN003 control cells (UPCP+shCtrl) that have taken up UPCP, HGADFN003 cells (UPCP+shBUBR1#3) that have taken up UPCP and have their endogenous BUBR1 knocked down show decreased expression of LaminB1, CyclinA2, and H3K9me3, indicating that reducing BUBR1 expression in HGADFN003 cells can inhibit UPCP-mediated upregulation of LaminB1, CyclinA2, and H3K9me3 expression. Figure 10 A).

[0198] 5) SA-β-gal senescence staining

[0199] HGADFN003 cells with UPCP uptake and endogenous BUBR1 knockdown, and HGADFN003 control cells with UPCP uptake were subjected to senescence staining using standard methods, referenced (Zhang, N., Ji, J., Zhou, D., Liu, X. & Zhang, Y. The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling. International Journal of Molecular Sciences 22, 849 (2021).). Results are as follows. Figure 10 As shown in B and 10C, Mock represents HGADFN003 cells transfected with shCtrl, UPCP represents HGADFN003 control cells that take up UPCP, and UPCP+shBUBR1#3 represents HGADFN003 cells that take up UPCP and have endogenous BUBR1 knocked down. Compared with HGADFN003 control cells that take up UPCP, the proportion of SA-β-gal positive cells in HGADFN003 cells that take up UPCP and have endogenous BUBR1 knocked down (UPCP+shBUBR1#3) is significantly increased, indicating that reducing BUBR1 expression can inhibit the reduction of SA-β-gal positive cells mediated by UPCP. Figure 10 B and Figure 10 C).

[0200] 6) Immunofluorescence assay

[0201] Immunofluorescence assay (using Ki67 antibody) was performed on HGADFN003 cells that had taken up UPCP and had their endogenous BUBR1 knocked down to detect Ki67 expression.

[0202] The results are as follows Figure 10 As shown in D (UPCP taken up with an N-terminal FITC-modified group) and 10E, Mock represents HGADFN003 cells transformed with shCtrl (shCtrl, indicated by Mock in the figure). Compared with HGADFN003 control cells that took up UPCP (FITC-UPCP or UPCP), the proportion of Ki67-positive cells in HGADFN003 cells that took up UPCP and had endogenous BUBR1 knocked down (FITC-UPCP+shBUBR1#3 or UPCP+shBUBR1#3) was significantly reduced, indicating that reducing BUBR1 expression can inhibit the increase of Ki67-positive cells in HGADFN003 cells mediated by UPCP. Figure 10 D and Figure 10 E).

[0203] 7) Chromosome karyotype analysis

[0204] Chromosomal karyotype analysis was performed on HGADFN003 cells that took up UPCP and had endogenous BUBR1 knocked down.

[0205] The results are as follows Figure 10 As shown in Figure F, Mock represents HGADFN003 cells transfected with shCtrl (shCtrl, denoted as Mock in the figure). Compared with HGADFN003 control cells (UPCP) that have taken up UPCP, the number of aneuploid cells in HGADFN003 cells that have taken up UPCP and have their endogenous BUBR1 knocked down (UPCP+shBUBR1#3) is increased, indicating that reducing BUBR1 expression can inhibit the UPCP-mediated reduction in the number of aneuploid cells in HGADFN003 cells. Figure 10 F).

[0206] The results above indicate that BUBR1 depletion can significantly antagonize the mitigation of UPCP-mediated cellular senescence phenotype, suggesting that UPCP restoration inhibits HGPS cell senescence by regulating BUBR1.

[0207] II. UPCP delays physiological cell senescence

[0208] Previous studies have shown that progerin accumulates in physiologically senescent cells, so the next step is to explore the effects of UPCP on normal physiological cell senescence.

[0209] AG09602 skin fibroblasts from a 92-year-old patient were cultured for 72 hours in medium containing 6 μM UPCP. AG09602 fibroblasts cultured in medium without UPCP served as a control.

[0210] Western blot was used to detect the expression levels of BUBR1, LaminB1, and CyclinA2 in skin fibroblasts of a 92-year-old patient after 72 hours of UPCP treatment.

[0211] The results are as follows Figure 11 As shown in Figure A, compared with the absence of UPCP, the addition of UPCP upregulated the expression of BUBR1, LaminB1, and CyclinA2 in the skin fibroblasts of a 92-year-old patient.

[0212] Simultaneously, skin fibroblasts from a 92-year-old elderly person, cultured in UPCP-containing medium for 72 hours, were subjected to SA-β-gal senescence staining to detect the effect of UPCP on physiological cell senescence, and Ki67 expression was detected by immunofluorescence assay. Fibroblasts AG09602 cultured in UPCP-free medium served as a control.

[0213] The results of aging staining showed that... Figure 11 As shown in B, UPCP can significantly reduce the proportion of SA-β-gal positive cells in physiologically senescent cells. Figure 11 B). Similarly, the results of the immunofluorescence experiment are as follows: Figure 11 C and Figure 11 As shown in D, UPCP (UPCP with an N-terminal FITC-modified group) can upregulate the expression of Ki67 in senescent cells.

[0214] The experimental data above indicate that UPCP can delay physiological cell senescence.

[0215] III. UPCP Security Testing

[0216] To further investigate whether UPCP has cytotoxic effects, IMR90 cells were cultured in medium containing 6 μM UPCP for 72 h. Cells cultured in medium without UPCP served as a control.

[0217] Western blot analysis was used to detect the expression of the proliferation marker Cyclin A2 in IMR90 cells after 72 h of culture. The results are as follows: Figure 12 As shown in Figure A, UPCP was found to have no significant effect on the expression of the IMR90 cell proliferation marker Cyclin A2. Figure 12 A).

[0218] Immunofluorescence assays were used to detect the expression of the Ki67 gene in IMR90 cells after 72 hours of culture. The results are as follows: Figure 12 As shown in B and 12C, UPCP had no effect on Ki67 expression in IMR90 cells. Figure 12 B and Figure 12 C).

[0219] Next, the Cell Counting Kit-8 (CCK-8) (APE k1018) was used to detect the effect of UPCP on the cell viability of IMR90 cells after culturing in medium containing different concentrations of UPCP for 72 h.

[0220] CCK-8 test results are as follows Figure 12 As shown in Figure D, different concentrations of UPCP had no effect on the growth of IMR90 cells compared to the control group. Figure 12 D).

[0221] The effects of different concentrations of UPCP on cell viability were statistically analyzed, and the results are as follows: Figure 12 As shown in E, different concentrations of UPCP did not affect the survival of IMR90 cells. Figure 12 E).

[0222] The above results indicate that UPCP has no significant toxic effect on cells.

[0223] IV. UPCP improves Lmna G609G / G609G Premature aging characteristics in premature aging mice and prolonging their lifespan.

[0224] 1. Administration of UPCP

[0225] male Lmna + / G609G Mice and female Lmna + / G609G Mice were crossbred, and the offspring mice were genotyped one week after birth. Lmna mice were selected at 14 days of age. G609G / G609G Premature aging mice were administered UPCP via intraperitoneal injection at a dose of 20 mg / kg every other day. The control group showed Lmna... G609G / G609G Premature aging mice were injected with the same amount of PBS in the same manner. The mice were continuously fed, their condition was observed, and their weight was measured regularly. The therapeutic effect of UPCP on premature aging mice was evaluated by comprehensively considering multiple indicators.

[0226] UPCP processing group (UPCP+ or Lmna) G609G / G609G +UPCP): In Lmna G609G / G609G Fourteen days after birth, mice were injected intraperitoneally with 20 mg / kg UPCP into Lmna via every other day. G609G / G609G In premature aging mice;

[0227] PBS-treated groups (UPCP- or Lmna) G609G / G609G ): Premature aging mice in the control group (Lmna) G609G / G609G Inject an equal volume of PBS.

[0228] Lmna + / +Group: ICR wild-type mice were injected with an equal volume of PBS.

[0229] 2. UPCP improves body weight in premature aging mice

[0230] After 12 weeks of injection of each substance in the groups obtained in 1 above, Lmna in male and female premature aging mice treated with UPCP was observed. G609G / G609G Individuals in the morphology group were larger than those in the PBS treatment group. Figure 13 A, D, Lmna in the diagram + / + (This refers to ICR wild-type mice).

[0231] The body weight of mice at different time points after injection was statistically analyzed, and male and female premature aging mice exhibited two different patterns of weight change. It was found that in the first 10 weeks after UPCP treatment, the UPCP-treated group (Lmna) showed significantly lower body weight. G609G / G609G +UPCP) and PBS-treated group (Lmna) G609G / G609G There was no significant difference in weight change among female premature aging mice treated with PBS after 10 weeks of treatment. However, the weight of female premature aging mice in the PBS-treated group decreased significantly, and the rate of weight loss in the UPCP-treated group was significantly slower than that in the PBS-treated group. Figure 13 B). It was also found that, compared with the PBS-treated group, the body weight of female premature aging mice did not increase significantly after 4 weeks of UPCP treatment, but after 12 weeks of UPCP treatment, the body weight of female premature aging mice in the UPCP-treated group was significantly higher than that in the PBS-treated group. Figure 13 C). In contrast, male premature aging mice experienced rapid weight gain followed by a stable weight gain in the early stages of UPCP treatment, while the UPCP treatment group (Lmna) showed a lower weight gain throughout the treatment period. G609G / G609G Male premature aging mice treated with UPCP consistently had a higher body weight than those treated with PBS (Lmna). G609G / G609G The body weight of male premature aging mice () Figure 13 E). Similarly, it was found that male premature aging mice treated with UPCP had significantly higher body weights at 4 and 12 weeks compared to the PBS-treated group. Figure 13 F).

[0232] The above-mentioned female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G Changes in body weight in mice, n = 6, 9, 9; male Lmna + / + , G609G / G609G and UPCP treatment for Lmna G609G / G609G The changes in mouse body weight, n = 11, 8, 6.

[0233] The above experimental results indicate that UPCP can significantly improve (enhance) premature aging Lmna. G609G / G609G Mouse body weight.

[0234] 3. UPCP on Lmna G609G / G609G Effects of premature aging mice on motor function

[0235] female Lmna + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G The mouse open field exercise test was conducted 12 weeks later to detect the effect of UPCP on Lmna. G609G / G609G The effects of premature aging on motor function in mice.

[0236] The results showed that, with Lmna + / + Compared to wild-type mice, Lmna G609G / G609G (PBS-treated group) premature aging mice showed significantly reduced motor function, while UPCP (Lmna) G609G / G609G +UPCP can precisely enhance Lmna G609G / G609G Motor ability of premature aging mice ( Figure 14 ).

[0237] This indicates that UPCP can alleviate motor dysfunction in premature aging mice to some extent.

[0238] 4. UPCP can prolong Lmna G609G / G609G Lifespan of premature aging mice

[0239] To further evaluate the effects of UPCP on Lmna G609G / G609G The therapeutic effects on premature aging mice were analyzed, and the survival time of mice in different groups was examined. This included studies on female Lmna mice. + / + Lmna G609G / G609G and UPCP treatment for Lmna G609G / G609G The survival time of Kaplan-Meier mice was statistically analyzed.

[0240] The results confirmed that UPCP can delay Lmna to some extent. G609G / G609G Survival time of premature aging mice ( Figure 15 Analysis revealed that Lmna in the PBS-treated group G609G / G609G The average lifespan of female premature aging mice was 101 days, with the longest lifespan being 107 days. However, after UPCP treatment, Lmna... G609G / G609G The average lifespan of female premature aging mice increased to 117 days, with the longest lifespan reaching 136 days. Figure 15 A). Similarly, in the PBS-treated group, Lmna G609G / G609G The average lifespan of male premature aging mice was 100 days, with the longest lifespan being 120 days. After UPCP treatment, Lmna... G609G / G609GThe average lifespan of male premature aging mice was extended to 116 days, with the longest lifespan reaching 135 days. Figure 15 B).

[0241] The above experimental results indicate that UPCP can prolong Lmna function to some extent. G609G / G609G Survival period of premature aging mice.

[0242] 5. UPCP adds Lmna G609G / G609G Subcutaneous fat in premature aging mice

[0243] Another clinical manifestation in HGPS patients is a reduction in subcutaneous adipose tissue. The next step is to test UPCP for Lmna. G609G / G609G Effects on skin tissue of prematurely aging mice.

[0244] Statistical analysis was performed on the skin HE staining (Absin, abs9217, performed according to the staining kit) and the fat layer of mouse skin tissue (Beyret E, Liao H K, Yamamoto M, et al. Single-dose CRISPR–Cas9 therapy extends lifespan of mice with Hutchinson–Gilford progeria syndrome[J]. Nature Medicine, 2019. Figure 2d) of mice obtained in UPCP treatment group and PBS treatment group 13 weeks after treatment. The results showed that, compared with wild-type Lmna + / + Compared to mouse skin tissue, Lmna G609G / G609G Premature aging mice showed a significant reduction or even disappearance of subcutaneous fat, along with a significant thinning of the epidermis and dermis; while Lmna after UPCP treatment... G609G / G609G Subcutaneous fat in premature aging mice was lower than that in the PBS-treated group, compared to Lmna G609G / G609G Premature aging mice have thicker fat layers, and Lmna was found to be more active after treatment. G609G / G609G The epidermis and dermis of the skin tissue of prematurely aging mice are wider. Figure 16 AB).

[0245] Therefore, the above results indicate that UPCP treatment can significantly improve Lmna G609G / G609G The reduction in subcutaneous fat and cortex in premature aging mice.

Claims

1. A polypeptide having an amino acid sequence as sequence 4 in the sequence listing.

2. The use of the polypeptide of claim 1 in the preparation of products having any of the following functions (A1)-A6): A1) Treatment for childhood progeria; A2) Delaying physiological aging in mammals or their cells; A3) Improves premature aging in mammals; A4) Inhibits HGPS cell senescence; A5) Promotes HGPS cell proliferation; A6) Inhibits aging caused by decreased BUBR1 expression.

3. The application according to claim 2, characterized in that: The improvement in premature aging in mammals is manifested in any of the following: B1) Improves the weight of prematurely aging animals; B2) Relieve motor dysfunction in prematurely aging animals; B3) Extend the lifespan of prematurely aging animals; B4) Improves the reduction of subcutaneous fat and / or cortex in premature aging animals.

4. A product comprising the polypeptide of claim 1.

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