Molecule for targeting senescent cells to stimulate endogenous immunity and use thereof

Abstract

The present invention relates to the field of biomedicine. Disclosed are a molecule for targeting senescent cells to stimulate endogenous immunity and use thereof. The drug provided by the present invention can mediate the interaction between senescent cells and immune cells, enhance the endogenous immune function, and achieve the goals of effectively clearing senescent cells and ameliorating aging-related diseases. A class of functional molecules targeting immune sensitization of senescent cells is provided. One end of the molecule targets membrane molecules of senescent cells, and labels the surface of senescent cells with glutamic acid, so as to mediate "coupling" between endogenous immune cells and senescent cells, thereby promoting the adhesion and clearance of senescent cells by immune cells. The molecule can regulate the immune microenvironment of aging tissues, and effectively reduce the number of senescence-associated β-galactosidase-positive senescent cells, while reversing fibrosis and improving the natural aging phenotype.

Description

Molecules and applications for targeting senescent cells to stimulate endogenous immunity Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a molecule for targeting senescent cells to stimulate endogenous immunity and its uses. Background Technology

[0002] It is estimated that my country's population aged 60 and above will exceed 400 million around 2035, accounting for more than 30% of the total population, entering a stage of severe aging. As the degree of population aging continues to deepen, achieving "healthy aging" has become a major social and scientific issue that urgently needs to be addressed in my country and even the world. Therefore, breakthroughs are urgently needed in the intervention of aging and related diseases to further improve the "healthy lifespan" of the elderly population and respond to the needs of the Healthy China strategy.

[0003] The accumulation of senescent cells in tissues can lead to diseases such as diabetes, osteoarthritis, liver and lung fibrosis, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), intestinal inflammation, and atherosclerosis. Immune clearance of senescent cells can slow aging. Multiple studies have shown the benefits of injecting transgenic or young immune cells to clear senescent cells from aging individuals, manifested in improved survival rates and histopathological improvements. These findings highlight the feasibility and potential of using immune strategies to clear senescent cells. However, exogenous immunotherapy carries the risks of cytokine release syndrome and neurotoxicity. In 2020, Nature reported on a chimeric antigen receptor T-cell (CAR-T) immunotherapy for selectively clearing senescent cells in mice. However, researchers also noted that a common complication of CAR-T therapy is cytokine release syndrome (also known as a cytokine storm). When this toxicity occurs, a strong T-cell response can lead to fever, affecting blood pressure and respiration. In this new study, the authors observed that high doses of anti-aging CAR-T cells did indeed induce cytokine release syndrome. Therefore, finding new methods to activate endogenous immune cells to reverse aging warrants further investigation.

[0004] The interaction and integration of the neuroendocrine and immune systems play a crucial role in the multi-system regulatory hierarchy of life. This interaction can mediate immune synaptic signaling through neurotransmitters (dopamine, acetylcholine, and γ-aminobutyric acid) and metabolic receptors present in the immune system. In one study, researchers compared metabolites in lymph nodes near immune sites with those in lymph nodes on the opposite side of a mouse's body after immune exposure to exogenous proteins. They found significant differences in the levels of metabolites associated with glutamate pathway activation in lymph nodes near immune response sites. The glutamate, alanine, and aspartate pathways are the strongest metabolic signatures distinguishing resting from activated immune sites. Glutamate is considered a neurotransmitter and immunotransmitter involved in signal transduction in the nervous and immune systems, and the expression and activation of metabolic glutamate receptors in immune cells may affect immune cell function and lifespan. Glutamate and its levels decline with age, and age is negatively and significantly correlated with metabolic signatures (serine and glutamate). Targeting glutamate receptors on immune cells may be a candidate drug for regulating glutamate receptor-mediated immunosensitization in the aging microenvironment. Therefore, strategies to promote the presentation of novel targets on the surface of the inflammatory microenvironment may enhance immune activity, thereby improving the efficacy of immunotherapy for aging. We are working to address this issue and determine whether immunotherapy targeting senescent cells can reverse chemical toxicity and tissue homeostasis in age-related diseases. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a molecule for targeting senescent cells to stimulate endogenous immunity and its applications. Specifically, this invention prepares a dual-ligand polypeptide that can act as a "mediator" and promoter to mediate the interaction between senescent cells and immune cells, enhance endogenous immune function, and achieve the goal of effectively clearing senescent cells and improving age-related diseases.

[0006] To achieve the objectives of this invention, the following technical solutions are provided:

[0007] This invention provides a molecule for targeting senescent cells to stimulate endogenous immunity.

[0008] The drug molecule contains at least two basic units.

[0009] One of the basic units is a unit with targeting functions for senescent cells, senescent tissues, or the senescent microenvironment.

[0010] Another basic unit is the unit used to stimulate endogenous immunity;

[0011] Alternatively, the molecule of the drug contains at least one basic unit, which is a unit capable of simultaneously targeting senescent cells, senescent tissues, or senescent microenvironments and stimulating endogenous immunity.

[0012] Specifically, this invention provides a molecule that induces immune activation in senescent cells. One end of the peptide is a ligand for urokinase-type plasminogen activator receptor (uPAR), which is widely expressed on the cell membrane of senescent cells, while the other end binds to a glutamate receptor, which is widely expressed on the cell membrane of immune cells. A chimeric polypeptide consisting of 40 amino acids is synthesized by adding polyglutamic acid to the amino terminus of the uPAR-specific ligand fragment. This polypeptide can target and bind to receptors on the surface of senescent cells and glutamate receptors, promoting the interaction between senescent cells and immune cells. By labeling senescent cells with glutamate, it activates glutamate receptors on immune cells, promoting their adhesion to immune cells and thus effectively clearing senescent cells.

[0013] There are no particular limitations on the target functional units in this invention, as long as they can be target functional molecular fragments for senescent cells / tissues / microenvironments.

[0014] Furthermore, the unit having the function of targeting senescent cells, senescent tissues, or senescent microenvironment is one or more of the following: antibodies targeting specific antigens on the surface of senescent cells, ligand molecules targeting receptors highly expressed on senescent cells, polypeptides targeting senescent cells, nucleic acid aptamers targeting senescent cells, polysaccharides targeting senescent cells, and molecules targeting the senescent microenvironment.

[0015] The polypeptides with specific targeting properties to senescent cells in this invention can be in linear or cyclic form.

[0016] Furthermore, the antibody targeting the specific antigen on the surface of senescent cells is an antibody against urokinase-type plasminogen activator receptor (uPAR).

[0017] The ligand molecule that targets the receptor highly expressed in senescent cells is urokinase-type plasminogen activator (uPA).

[0018] The polypeptide targeting senescent cells is a urokinase-type plasminogen activator (PURA)-binding domain polypeptide. In some embodiments of this invention, animal experiments were first conducted using mice. Therefore, when conducting experiments with mice, the urokinase-type PURA-binding domain polypeptide was CQNGGVCVSYKYFSRIRRCSCPRK (as shown in SEQ ID NO:1), i.e., a mouse-derived sequence. However, the human urokinase-type PURA-binding domain polypeptide differs from this sequence. Therefore, when conducting experiments targeting humans, the urokinase-type PURA-binding domain polypeptide was LNGGTCVSNKYFSNIHWCNCPKKF (as shown in SEQ ID NO:2), i.e., a human-derived sequence. Of course, the sequences mentioned above in this invention can be all or part of the sequence, such as SNKYFS (as shown in SEQ ID NO:3), VSNKYFSNI (as shown in SEQ ID NO:4), and VSNKYFSNIHW (as shown in SEQ ID NO:5), as long as they satisfy the targeting function.

[0019] Peptides can be linear molecules or cyclic peptides.

[0020] Nucleic acid aptamers that target senescent cells are nucleic acid aptamers with specific targeting properties for senescent cells;

[0021] The polysaccharide that targets senescent cells is β-galactose, which targets β-galactosidase, a marker of senescent cells.

[0022] Molecules that target the aging microenvironment are molecules that target specific microenvironments related to aging.

[0023] Preferably, the unit for stimulating endogenous immunity is a molecule capable of activating metabolic glutamate receptors or capable of stimulating immune cells in aging tissues or the aging microenvironment.

[0024] Furthermore, the molecule capable of activating metabolic glutamate receptors is one or more of a polyglutamate repeating unit and a glutamate agonist.

[0025] The immune cells are natural killer cells (NK cells), macrophages, T cells, or dendritic cells.

[0026] Furthermore, the targeting unit of the molecule used to target senescent cells and stimulate endogenous immunity is selected as a urokinase-type plasminogen activator binding domain polypeptide, and the unit used to stimulate endogenous immunity is selected as a polyglutamic acid repeat unit. In this case, the murine amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. x CQNGGVCVSYKYFSRIRRCSCPRK, where x is any integer.

[0027] A molecule designed to target senescent cells and stimulate endogenous immunity; its human amino acid sequence is E. y LNGGTCVSNKYFSNIHWCNCPKKF is a sequence of all or part of the sequence, where y is any integer.

[0028] Furthermore, the molecule used to target senescent cells and stimulate endogenous immunity is E. y SNKYFS, E y VSNKYFSNI, E y One of the VSNKYFSNIHW.

[0029] If a targeted peptide molecule contains one or more glutamates, it may simultaneously fulfill both dual targeting and endogenous immune stimulation functions, independently achieving the purpose of this invention. However, targeted peptide molecules are often affected by the hydrophilicity or hydrophobicity of different glutamates, which may influence the targeting affinity of the targeted peptide.

[0030] The targeting portion of the polypeptide molecule is responsible for binding to membrane proteins highly expressed on the surface of senescent cell membranes, preventing off-target effects. Therefore, due to space limitations, not all of them are listed exhaustively. However, other targeting molecules not described in this invention can also play the same role. The immune-stimulating portion can be a repeating unit containing polyglutamate (En, n≥1) or a glutamate receptor activator. The immune-stimulating portion is responsible for activating endogenous immune cells. Other glutamate receptors that can activate class I metabolic glutamate receptors, not described in detail in this invention, can also play the same role.

[0031] Furthermore, in our experiments, we found that the mouse-derived polypeptide molecules shown in SEQ ID NO:1 above have low solubility, and increasing the amount of glutamate can increase their solubility. However, the amount of glutamate is negatively correlated with the targeting of the molecule. Therefore, considering all factors, 8≤x≤24.

[0032] The human-derived peptide molecules disclosed above have relatively good solubility, so there is no need to consider increasing glutamate to improve their solubility. Instead, we should focus on the molecular targeting specificity and the effect of stimulating immunity. Taking all factors into account, 1≤y≤24.

[0033] In this invention, the two functional regions (targeting + immunosensitization) that target senescent cells to stimulate endogenous immunity can be achieved by two different molecular units (targeting functional domain + immunosensitization functional domain), such as uPA-polyglutamic acid crosslinking molecules; or they can be combined into one, that is, one functional unit has both of these functions (targeting + immunosensitization), such as huPA.

[0034] Specifically, our further research revealed that the mouse-derived uPA sequence currently struggles to simultaneously achieve both targeting and stimulation functions in experiments. In contrast, the human-derived huPA sequence demonstrated in experiments that it can simultaneously target senescent cells and stimulate endogenous immunity.

[0035] Therefore, further targeted stimulation of senescent cells with endogenous immune molecules may contain only one basic unit, which is a polypeptide molecule with an amino acid sequence of all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

[0036] Furthermore, the amino acid sequence of the polypeptide molecule can be one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, or VSNKYFSNIHW.

[0037] The present invention also provides the use of the described molecule in the preparation of drugs or health products for reversing natural aging, and can also be used in drugs for reversing age-related diseases.

[0038] When applied, the administration methods include intravenous administration, interventional administration to tissue lesions, oral administration, nasal inhalation administration, or intraperitoneal injection.

[0039] The molecule for targeting senescent cells and stimulating immunity provided by this invention has been experimentally verified to specifically target senescent cells, stimulate immune cells, including NK cells, and eliminate senescent cells. Therefore, in principle, this molecule for targeting senescent cells and stimulating immunity can be used to prepare drugs suitable for treating all fibrosis and age-related diseases, including Alzheimer's disease, Parkinson's disease, atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by some genotoxic chemotherapy), tumorigenesis, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, liver fibrosis, kidney fibrosis, pulmonary fibrosis, myocardial fibrosis and other organ fibrosis diseases, chronic obstructive pulmonary disease, intestinal inflammation, diabetes, osteoarthritis, osteoporosis, macular degeneration, ovarian aging, natural aging including skin aging, and synergistic tumor treatment.

[0040] The present invention also provides the use of the above-mentioned molecules that target senescent cells to stimulate immunity in the preparation of drugs for clearing senescent cells.

[0041] In addition, the present invention also provides a senescent cell clearing drug, the drug comprising the above-mentioned molecules that target senescent cells and stimulate immunity.

[0042] The senescent cell clearing drug contains, in addition to the senescent cell-stimulating immune molecules of the present invention, necessary excipients, and may also contain other therapeutic drugs.

[0043] Furthermore, the senescent cell clearing drug can deliver the molecules that target senescent cells to stimulate immunity via oral, intravenous, intralesional, or intraperitoneal injection.

[0044] The present invention also provides a cosmetic composition that can delay and improve skin aging, the composition comprising the above-mentioned molecules that target aging cells and stimulate immunity.

[0045] The present invention also provides a health product composition for delaying aging, wherein the composition contains the above-mentioned molecules that target aging cells and stimulate immunity.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention provides a class of functional molecules that target senescent cells to stimulate immunity. One end of this molecule targets senescent cell membrane molecules and labels the surface of senescent cells with glutamate, mediating a "coupling" between endogenous immune cells and senescent cells, promoting the adhesion and clearance of senescent cells by immune cells. This molecule can regulate the immune microenvironment of senescent tissues, effectively reducing the number of senescence-associated β-galactosidase-positive senescent cells, while reversing fibrosis and improving the natural aging phenotype, thus treating and improving cellular senescence-related diseases. Attached Figure Description

[0048] Figure 1 shows the uPAR expression in the replicated senescence and chemotherapy-induced cellular senescence models in Example 1. a represents the SA-β-gal staining of expanded mouse embryonic fibroblasts (MEF) cells from generations 2, 7, and 9, and the detection of uPAR protein levels in MEF cells of different generations. Blue-green indicates positive staining. b represents the SA-β-gal staining of LLC cells in the quiescent, proliferative, and bleomycin (BLM) treatment phases, and the detection of uPAR protein levels.

[0049] Figure 2 shows the E16-uPA in Example 1. 24 It specifically binds to uPAR on the membranes of senescent cells. a, Chimeric polypeptide E16-uPA 24 b. Dynamic binding sensing diagram of uPAR recombinant protein. Flow cytometry analysis of the target peptides binding on the surface of young and senescent MEF cells. As shown in the figure, the rightward shift of the peak indicates that the chimeric peptide molecules selectively bind to the surface of uPAR-positive senescent cells, while not binding to uPAR-negative young cells.

[0050] Figure 3 shows the co-localization of chimeric peptides and uPAR immunofluorescence in senescent sites in liver tissue sections of mice with liver fibrosis, as displayed by co-focused immunofluorescence staining in Example 1.

[0051] Figure 4 shows the mass cytometry analysis of immune cells in carbon tetrachloride (CCl4)-induced liver fibrosis in Example 2. a) E16-uPA in CCl4-induced liver fibrosis. 24 The treatment diagram shows the normal control group, the CCl4 model group, and the E16-uPA group. 24 Mass cytometry visualization of t-SNE data of immune cells in liver tissue. b, normal control group, CCl4 model group and E16-uPA group. 24 The proportion of different immune cell subsets in the group. T-SNE represents t-distributed random neighbor embedding. DNT represents double-negative T cells, CD4-CD8-TCRb. + Cells. DPT represents double-positive T cells, CD4 + CD8 + TCRb + cell.

[0052] Figure 5 shows E16-uPA in Example 3. 24 Stimulation activates NK cells. a. E16-uPA was quantified by flow cytometry. 24 Incubated LLC senescent cells with CD45 + CD3 in cell co-culture system + CD4-T cells and CD3-NK1.1 + The proportion of CD107a-positive NK cells. CD45 cells incubated with solvent. + Cells shown are a negative control. CD45 cells incubated with PMA / ion + Cells served as a positive control. b, purified NK cells in E16-uPA... 24 The release of interferon-gamma after incubation exhibits cytotoxicity.

[0053] Figure 6 shows the E16-uPA in Example 3. 24 Activates NK cells to specifically kill senescent cells. (a) Compared with different concentrations of E16-uPA... 24 NK cell cytotoxicity analysis after co-culturing young and senescent MEF cells with pre-incubated cells. b, Purified NK cells with different concentrations of uPA. 24 E16 and E16-uPA 24 Cytotoxicity of pre-incubated senescent MEF cells after co-culture.

[0054] Figure 7 shows E16-uPA in Example 4. 24 It exerts its anti-fibrotic effect by clearing senescent cells in CCl4-induced liver fibrosis. a, b, compare the severity of liver fibrosis in different treatment groups at 0, 6, and 12 weeks of induction based on the mean echogenicity of the liver. c, compare the groups (healthy control group, D+Q group, PBS group, uPA group) using scatter histograms.24 Group, E16 group and E16-uPA 24 The statistical differences in Sirius red staining of liver tissue from each group were used to determine the treatment efficacy. (d) The groups (healthy control group, D+Q group, PBS group, uPA group) were compared. 24 Group, E16 group and E16-uPA 24 Scatter histogram comparing SA-β-gal staining of liver tissue from different groups. e, healthy control group, D+Q group, PBS group, uPA group 24 Group, E16 group and E16-uPA 24 Quantitative analysis of the NK cell marker CD161c in paraffin sections of liver tissue in the group.

[0055] Figure 8 shows E16-uPA in Example 5. 24 It can eliminate senescent cells and alleviate lung damage. a) For the first 5 days of treatment, mice were administered BLM (2 mg / kg) or saline (normal control group) via intratracheal injection. Mice with lung injury were then intravenously injected with E16-uPA for two consecutive weeks. 24 (50 mg / kg) or physiological saline (carrier group), 3 times a week, scatter histograms of statistical comparison of Masson staining of lung tissue in the three groups (n=4). b, Scatter histogram showing healthy group, control group or E16-uPA 24 Comparison of SA-β-Gal staining in lung tissue of the treatment group. c, healthy group, control group or E16-uPA 24 Quantitative analysis of the NK cell marker CD161c in paraffin sections of lung tissue from the treatment group. (d. Healthy group, control group, or E16-uPA) 24 Expression of aging markers in lung tissue of group (n=3). E16-uPA was analyzed using the Mantel-Cox log-rank test. 24 Kaplan-Meier survival curves of lung-injured mice treated with saline or physiological saline (n=10).

[0056] Figure 9 shows E16-uPA in Example 5. 24 NK cell therapy for pulmonary fibrosis in mice. a, b, control group and E16-uPA. 24 CD45 in the treatment group + Quantitative analysis of the percentage of NK cells in lung immune cells and flow cytometry cell count.

[0057] Figure 10 shows the E16-uPA in Example 6. 24 Relief of age-related phenotypes in naturally aging mice. a, Schematic diagram of the drug administration process for 22-month-old C57BL / 6J mice: E16-uPA administered three times weekly. 24 For two consecutive months. b, aged mouse control group, E 16 -uPA 24Forelimb grip strength test of group D+Q (n=8). c, Rotary fatigue test of three groups of aged mice (n=8). d, f, Young mice vs. aged mice (control), E16-uPA 24 Representative images and quantitative analysis of SA-β-gal staining in liver tissue sections from the D+Q treatment group (n=3); liver tissue cell nuclei were counterstained red. e, g, young mice (n=5) and aged mice as controls, E16-uPA 24 Representative images and quantitative analysis of SA-β-gal staining of kidney tissue sections from the D+Q treatment group (n=8); no nuclear counterstaining was performed on the kidney tissues. h, i, young mice and aged mice as controls, E16-uPA 24 Representative images and quantitative analysis of SA-β-gal staining in inguinal white adipose tissue (iWAT) of the D+Q treatment group (n=6). j–n, young mice (n=5) and aged mice controls, E16-uPA. 24 The expression levels of SASP factors IL-1, IL-6, CXCL1, TNF, and MMP-1 in the serum of the D+Q treatment group (n=8) were compared with those in the D+Q treatment group. Figure 11 shows the expression levels of Ey-huPA in Example 7. 24 Targeted sensitization of human senescent cells triggers NK cell killing. a, compared with different concentrations of E1-huPA 24 NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with pre-incubated cells. b, Comparison with cells cultured with different concentrations of E2-huPA. 24 NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with different concentrations of E4-huPA. 24 Cytotoxicity analysis of NK92 cells co-cultured with pre-incubated young and senescent IMR90 cells. (d, compared with cells cultured with different concentrations of E8-huPA) 24 NK92 cytotoxicity analysis after co-culturing young and senescent IMR90 cells with different concentrations of E16-huPA. 24 NK92 cytotoxicity analysis after co-culturing pre-incubated young and senescent IMR90 cells. f, Ey-huPA 24 Quantitative analysis of IFN-γ in cell culture supernatant after co-incubation with NK92. (g, different Ey-huPA) 24 Flow cytometry analysis of binding affinity to young IMR90 cells. h, different Ey-huPA 24 Flow cytometry quantitative analysis of binding ability with senescent IMR90 cells. i. Flow cytometry detection of senescent and young cells in huPA. 24Quantitative statistics on cell mortality after co-treatment with NK92 cells.

[0058] Figure 12 illustrates the NK cell killing effect triggered by E4-huPAz targeting human senescent cells in Example 8. a) NK92 cytotoxicity analysis after co-culturing with young and senescent IMR90 cells pre-incubated with different concentrations of E4-huPA6. b) NK92 cytotoxicity analysis after co-culturing with young and senescent IMR90 cells pre-incubated with different concentrations of E4-huPA9. c) NK92 cytotoxicity analysis after co-culturing with different concentrations of E4-huPA9. 11 d. Quantitative analysis of IFN-γ in cell culture supernatant after co-incubation of pre-incubated young and aged IMR90 cells. e. Flow cytometry quantitative analysis of the binding affinity of different E4-huPAz to aged IMR90 cells. f. Flow cytometry quantitative analysis of the binding affinity of different E4-huPAz to young IMR90 cells.

[0059] Figure 13 shows E16-uPA in Example 9. 24 To alleviate ovarian aging in a chemotherapy-induced aging mouse model. a) ELISA quantitative analysis of serum levels of inhibin B, estradiol, follicle-stimulating hormone, and luteinizing hormone in mice. b) Quantitative analysis of ovarian-to-body weight ratio in mice. c) Quantitative analysis of the number of ovaries in a single ovary in mice.

[0060] Figure 14 shows E16-uPA in Example 9. 24 To alleviate ovarian aging in a naturally aging mouse model, E16-uPA was injected via the tail vein into mice every other day. 24 Analysis of estrous cycles in mice 45 days after drug administration. b. Mice were intraperitoneally injected with E16-uPA every other day. 24 c) Analysis of estrous cycles in mice 45 days after drug administration. d) Analysis of estrous cycles in mice 45 days after administration of saline injected via the tail vein every other day.

[0061] Figure 15 shows the E16-uPA in Example 10. 24 To alleviate skin aging in a naturally aging mouse model. a) Mice were subcutaneously injected with physiological saline and 5 mg / kg of E16-uPA. 24 15 mg / kg of E16-uPA 24a) HE and Masson staining images of mouse skin at the drug administration site 21 days later. b) P16 and uPAR expression in mouse skin tissue detected by Western blotting. c) Quantitative statistics of DEJ convolution at the dermal-epidermal junction in mice. d) Quantitative statistics of dermal thickness in mice. e) Quantitative statistics of epidermal thickness in mice. f) Quantitative statistics of collagen in mouse Masson staining images.

[0062] Figure 16 shows the E16-uPA in Example 11. 24 A mouse model of dry macular degeneration. a) Representative images of the extraretinal nucleus layer (ONL) and retinal pigment epithelium (RPE) cell layers in mouse eyes stained with hematoxylin and eosin (HE), and immunofluorescence staining of retinal p16. b) Quantitative statistics of ONL thickness in mice. c) Quantitative statistics of RPE layer thickness in mice. d) Quantitative statistics of p16 immunofluorescence intensity in mice. e) Expression of p16, p53, and uPAR in the mouse retina as detected by Western blotting.

[0063] Figure 17 shows the E16-uPA in Example 12. 24 Delaying tumor progression and reducing chemotherapy toxicity. a) Quantitative statistical analysis of tumor volume in mice. b) Quantitative statistical analysis of senescence-related β-galactosidase-positive areas in mouse tumor tissue. c) Quantitative statistical analysis of pulmonary fibrosis areas in mice. d) Statistical analysis of mouse survival curves. e) Quantitative statistical analysis of CD161c staining intensity in mouse tumor tissue.

[0064] Figure 18 shows the E16-uPA in Example 13. 24 Results showing improvement in liver and kidney function in naturally aging mice. a–c, young mice, aged control group, E16-uPA. 24 Immunoblotting and quantitative analysis of uPAR and p16 proteins in kidney tissues of the treatment group and the D+Q treatment group (n=3). d–f, young mice, aged control group, E16-uPAR 24 Immunoblotting and quantitative analysis of uPAR and p16 proteins in liver tissues of the treatment group and the D+Q treatment group (n=3). g–i, quantitative analysis of serum liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) levels (n=6). j–l, quantitative analysis of serum renal function indicators creatinine (CREA), uric acid (UA), and urea (BUN) levels (n=6). m, young mice, aged mice, and E16-uPAR... 24 Heatmap analysis of aging-related genes in the kidneys of aged mice (n=3).

[0065] Figure 19 shows the E16-uPA in Example 13. 24Improved the expression of aging-related genes in multiple tissues of naturally aging mice. (a) Young mice, aged mice, and E16-uPA... 24 Heatmap analysis of aging-related genes in the liver of aged mice (n=3). b, young mice, aged mice, and E16-uPA. 24 Heatmap analysis of aging-related genes in lung tissue of aged mice (n=3).

[0066] Figure 20 shows E1-uPA in Example 14. 24 (E) and E16-uPA 24 (E-16) Echocardiographic results of improved cardiac function in a cardiac fibrosis model. a, Postoperative 7 days, groups (sham surgery group, cardiac fibrosis group, cardiac fibrosis + E1-uPA) 24 Group, cardiac fibrosis + E16-uPA 24 a) Representative echocardiographic images of mice in each group. b) Quantitative analysis of left ventricular ejection fraction (EF) in each group (n=4). c) Quantitative analysis of left ventricular fractional shortening (FS) in each group (n=4). d) Quantitative analysis of left ventricular end-systolic diameters (LVIDs) in each group (n=4). e) Quantitative analysis of left ventricular end-diastolic diameters (LVIDd) in each group (n=4). Detailed Implementation

[0067] Example 1

[0068] To verify the molecular effects of this invention, animal experiments were first conducted. Mice, a common laboratory animal, were selected as the research subject. Therefore, the mouse senescent cell-targeting chimeric peptide (E) was chosen. x CQNGGVCVSYKYFSRIRRCSCPRK (x is any integer) reverses chemical toxicity and tissue homeostasis associated with age-related diseases by stimulating immune cells through targeting senescent cells.

[0069] As mentioned earlier, the polypeptide molecule in mice has poor solubility. Therefore, in the examples, experiments were conducted by linking more glutamic acid molecules. In the following studies on mice, the examples of the present invention mainly use 8, 16, and 24 glutamic acid molecules as representatives. This does not mean that other numbers of glutamic acid molecules cannot achieve the corresponding function.

[0070] Example of polypeptide sequence abbreviation: E 16 CQNGGVCVSYKYFSRIRRCSCPRK(E16-uPA 24 ),E8CQNGGVCVSYKYFSRIRRCSCPRK(E8-uPA 24 E 24 CQNGGVCVSYKYFSRIRRCSCPRK(E24-uPA24 ).

[0071] (1) uPAR is highly expressed in senescent cells

[0072] To establish a suitable cell senescence model, for replication-induced senescence, primary-derived first-generation MEF cells were cultured using normal cell passages until they lost their proliferative capacity and became senescent. MEF cells typically enter senescence after approximately 7 cell passages or 10 population doublings. For bleomycin-induced senescent Lewis lung carcinoma (LLC) cells, LLC cells were treated with fresh BLM medium at a concentration of 30 μg / mL for approximately 24 to 48 hours, with the medium being replaced with fresh medium, until the cells ceased proliferation and entered senescence. The results showed that MEF cells began to senescent at the 7th generation and had essentially entered senescence by the 9th generation, ceasing proliferation. uPAR expression gradually increased with increasing MEF cell passage number, with low-generation MEF cells showing almost no uPAR expression (Figure 1a). BLM treatment induced senescence in mouse LLC cells, while serum starvation induced mouse LLC cells to enter a quiescent phase served as a control. Five days after drug treatment, most cells were successfully induced to enter the senescence phase. Quiescent LLC cells expressed almost no uPAR, but after 24 hours of drug treatment to induce senescence, the expression of uPAR on the cell surface gradually increased over time (Figure 1b). This indicates that cells exhibit high uPAR expression after senescence induced by chemotherapy or replication and passage.

[0073] (2)E16-uPA 24 Specific binding of uPAR

[0074] Based on surface plasmon resonance (SPR) to detect the affinity of peptides for uPAR, a commercially available His-tagged recombinant uPAR protein was first immobilized on a CM5 sensor chip with a carboxymethyl dextran matrix covalently linked to a gold surface. Subsequently, a series of E16-uPAR proteins at different dilution ratios were... 24 Injected onto the sensor surface, the chimeric polypeptide exhibited concentration-dependent binding activity with the uPAR protein. Based on the results, E16-uPAR... 24 Suitable for kinetic fitting calculations. As shown in the figure, the response signal (Response, RU) increases with increasing sample concentration, and E16-uPA can be calculated accordingly. 24 The KD value is 7.310 × 10 -8 M (Fig. 2a). Chimeric polypeptide E16-uPA 24 It has a relatively high affinity for uPAR.

[0075] To establish a suitable cellular senescence model, we induced MEF cell replication and senescence through cell expansion (Figure 1a). Flow cytometry results also showed the presence of the chimeric peptide E16-uPA. 24 The number of senescent MEF cells was significantly higher than that of young MEF cells (Fig. 2b).

[0076] To determine the chimeric peptide E16-uPA 24 Specific targeting of aging tissues, further confirmed by tissue immunofluorescence staining, revealed E16-uPA. 24 Co-localization with uPAR-positive cells. Normal mice and CCl4-treated mice were intravenously injected with E16-uPAR conjugated with 5-FAM. 24 After 4 hours of metabolism, liver tissue was dissected and frozen sections were prepared using E16 and stained with uPAR antibody. Colocalization analysis of the liver tissue 4 hours after metabolism revealed (Figure 3) E16-uPAR. 24 The fluorescence (green) of the chimeric polypeptide E16-uPA clearly overlaps with the fluorescence (red) of the uPAR antibody. This indicates that the chimeric polypeptide E16-uPAR... 24 It can be targeted to senescent cell membranes and senescent tissue sites by combining with uPAR.

[0077] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK all found that tail vein injection of uPAR-targeting peptides can significantly target senescent cells.

[0078] Example 2

[0079] Balance the immune microenvironment in liver fibrosis tissue.

[0080] Liver fibrosis was induced in C57BL / 6N mice by intraperitoneal injection of 1 ml / kg CCl4 twice weekly. After six weeks, mice with similar degrees of liver fibrosis were randomly divided into a control group and a treatment group. Subsequently, the mice were injected via tail vein with the peptide E16-uPA. 24 Mice were given 50 mg / kg of PBS three times a week for six weeks, while continuing CCl4 treatment at the same dose and intervals. Normal liver tissue and PBS or E16-uPA were collected from mice after treatment. 24 Post-treatment fibrotic tissue was analyzed, and immune cells were characterized by mass cytometry (Figure 4). Analysis revealed approximately 12 distinct clusters (Figure 4b). t-SNE plots showed the locational clustering of large immune cell subsets: dendritic cells (DCs), macrophages, B cells, eosinophils, basophils, neutrophils, and CD4+. + T cells, CD4 + Treg cells, CD8+ T cells, CD4 - CD8 - TCRb + Cells, NK cells and CD4 + CD8 + TCRb + Cells (Fig. 4a). Eosinophils, basophils, NK cells, and CD4+. + T cells and other large cell subsets in the CCl4 model group and E16-uPA 24 The numerical changes between the treatment groups were statistically significant. We found that, compared with the CCl4 group, the treatment groups had a decreased proportion of pro-inflammatory granulocytes (eosinophils), a decreasing trend in neutrophils, and a decrease in cytotoxic lymphocytes (NK cells and CD4+). + The proportion of T cells increased significantly (Figure 4b). These findings indicate that E16-uPA... 24 It can regulate the microenvironment of organ aging by enhancing the cytotoxic killing effect of immune cells.

[0081] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were all found to regulate the organ aging microenvironment by enhancing the cytotoxic killing effect of immune cells.

[0082] Example 3

[0083] It sensitizes senescent cells, triggering NK cell killing.

[0084] Senescent LLC cells from Example 1 were cultured at 4000 cells / well using E16-uPA. 24 After 0.5 h of pre-incubation, CD45 isolated from mouse spleen + Immune cells were co-cultured for 6 hours. Flow cytometry analysis showed that, compared with T cells, CD3-NK1.1... + NK cells exposed to E16-uPA 24 Subsequently, the degranulation marker CD107a was significantly induced (Fig. 5a), and CD3-NK1.1 + The expression of CD107a, a marker of NK cell degranulation, was significantly increased, by approximately 22.12%. This indicates that NK cells are responding to E16-uPA. 24 The main effector cells. When the targeting peptide uPA... 24 Polypeptide E16 and chimeric polypeptide E16-uPA 24 After co-incubation with NK cells for 48 hours, E16-uPA 24It can significantly stimulate the production of interferon-γ, while E16 has only a weak activating effect (Figure 5b). Next, young MEF cells and senescent MEF cells were compared with different concentrations of E16-uPA. 24 After incubation, the cells were co-cultured with NK cells isolated and purified from the spleen. Results showed that the NK cells eliminated the cytotoxicity of senescent cells over a broad E16-uPA spectrum. 24 Improvement was observed at certain drug concentrations, but no such effect was observed in young cells (Figure 6a). Furthermore, compared to E16 and uPA... 16 Only E16-uPA 24 It can significantly promote the cytotoxic effect of NK cells against senescent cells (Figure 6b). E16 can also activate the cytotoxic killing effect of NK cells to some extent. The above results indicate that E16-uPA 24 It can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0085] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK can all effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0086] Example 4

[0087] Recruit NK cells to eliminate senescent cells and reduce fibrosis in a liver fibrosis model.

[0088] Liver fibrosis was induced in C57BL / 6N mice by intraperitoneal injection of 1 ml / kg CCl4 twice weekly. After six weeks, mice with similar degrees of liver fibrosis were randomly divided into a model vector group and a treatment group. Subsequently, the treatment group was injected with the peptide E16-uPA via the tail vein. 24 (50 mg / kg), polypeptide uPA 24 Mice receiving PBS (50 mg / kg), peptide E16 (50 mg / kg), D+Q (50 mg / kg dasatinib and 5 mg / kg quercetin), and the model vector group were treated three times a week for six weeks, while continuing CCl4 treatment at the same dose and intervals. Liver ultrasound and histopathological sections were performed on normal mice and treated mice at weeks 0, 6, and 12. Imaging results showed E16-uPA 24 The single treatment achieved the same effect as the synergistic treatment with D+Q anti-aging agents, using E16-uPA. 24Both treatment with anti-aging agents reversed liver fibrosis after 6 weeks, characterized by reduced and more homogeneous echogenicity (Fig. 7a, b); and single-drug E16-uPA was also assessed by SA-β-Gal and Sirius red staining. 24 Liver samples obtained after D+Q treatment showed fewer senescent cells and less fibrosis (Fig. 7c, d). Immunohistochemical staining of mouse liver tissue sections for the NK cell marker CD161c revealed that CD161c was present in E16-uPA... 24 The treatment group showed significantly increased staining intensity, indicating marked NK cell infiltration in the liver fibrosis area (Fig. 7e). This suggests that E16-uPA... 24 It can effectively eliminate senescent cells in the fibrotic liver of mice.

[0089] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were all found to effectively eliminate senescent cells in the fibrotic liver of mice.

[0090] Example 5

[0091] Recruit NK cells to eliminate senescent cells and reduce fibrosis in a mouse lung injury model.

[0092] Male mice (8-12 weeks old) were administered bleomycin (Selleck, S1214) via intratracheal injection at a dose of 2 mg / kg. Following inoculation, mice were randomly divided into a control group and a treatment group based on their body weight changes over 5 days. The treatment group received E16-uPA intravenously three times weekly starting on day 5. 24 (50 mg / kg) or physiological saline, treatment lasted for 2 weeks. Lung tissue was collected from treated mice for Masson staining, β-galactosidase staining, and CD161c immunohistochemistry and other pathological staining. As shown in Figure 8, after E16-uPA... 24 Following treatment, the area of ​​pulmonary collagen fiber staining and the percentage of SA-β-gal positive cells were significantly reduced, while NK cell infiltration increased (Fig. 8a, b, c). Simultaneously, the mRNA levels of aging-related markers such as Col1a1, Serpine1, and P21 genes were also significantly decreased (Fig. 8d). Since there was a statistically significant increase in survival compared to vehicle-injected mice, it is concluded that removing senescence and inhibiting fibrosis are beneficial for survival (Fig. 8e). These results indicate that E16-uPA... 24 Treatment of activated NK cells in the lungs can attenuate the accumulation of senescent cells during bleomycin-induced pulmonary fibrosis, thereby producing an anti-fibrotic effect and improving survival rate. E16-uPA 24 Treatment can reduce the secretion of aging inflammatory factors to a certain extent.

[0093] E16-uPA 24 NK cell therapy for pulmonary fibrosis in mice. a, b, control group and E16-uPA. 24 CD45 in the treatment group + Quantitative analysis of the percentage of NK cells in lung immune cells and flow cytometry cell counting. In the quantitative results of flow cytometry, E16-uPA... 24 The proportion of NK cells in the lungs of the treatment group was also significantly higher than that of the solvent group (Figure 9), indicating that E16-uPA 24 Treatment can effectively recruit NK cells to aggregate in the lung injury area.

[0094] Repeat the above experimental method, and use respectively

[0095] E8CQNGGVCVSYKYFSRIRRCSCPRK and

[0096] E 24 Treatment with CQNGGVCVSYKYFSRIRRCSCPRK can effectively recruit NK cell aggregation in the lung injury area.

[0097] Example 6

[0098] Eliminate senescent cells in naturally aging mice.

[0099] To further investigate the effects of chimeric peptides on naturally occurring senescent cells in aged mice, we used E16-uPA on 22-month-old C57 mice. 24 (50 mg / kg), D+Q (50 mg / kg dasatinib and 5 mg / kg quercetin) or blank solution, injected three times weekly for 2 months, followed by assessment by SA-β-Gal staining for chimeric peptide E16-uPA. 24 Both treatment and the combined D+Q treatment significantly reduced SA-β-gal activity in the liver, kidney, and inguinal adipose tissue of aged mice, suggesting a significant reduction in senescent cells in these regions. These results indicate that the chimeric peptide E16-uPA... 24 It can systematically improve the aging pathological characteristics of naturally aging mice.

[0100] The specific experimental methods and steps are as follows:

[0101] We selected 22-month-old C57BL / 6J mice as experimental subjects. The mice were randomly divided into a control group and an E16-uPA control group. 24The treatment group (50 mg / kg, administered via tail vein three times a week) and the positive control D+Q treatment group (dasatinib 50 mg / kg + quercetin 5 mg / kg, administered via tail vein three times a week) were administered continuously for 2 months.

[0102] After the dosing cycle ended, a series of functional tests were performed on the mice. The results showed that E16-uPA... 24 The forelimb grip strength of the mice in the group was significantly enhanced (Fig. 10b), and their exercise endurance in the rotundus fatigue test was also significantly improved (Fig. 10c), suggesting that E16-uPA... 24 It can improve muscle function in aged mice.

[0103] The levels of senescent cells in different tissues were further detected by SA-β-gal staining. The results showed that, compared with the control group, E16-uPA... 24 The proportion of senescent cells in the liver and kidney tissues of the treated mice was significantly reduced (Fig. 10d–g). Cell nuclei in liver tissue sections were counterstained red, while kidney tissue sections were not counterstained. Detection of inguinal white adipose tissue (iWAT) also showed that E16-uPA... 24 The burden of senescent cells in the group was significantly reduced (Fig. 10h–i).

[0104] Furthermore, detection of serum inflammatory factor levels showed that E16-uPA 24 It can effectively reduce the expression levels of multiple SASP factors in the serum of naturally aging mice, including IL-1, IL-6, CXCL1, TNF and MMP-1 (Figure 10j–n).

[0105] In summary, E16-uPA 24 It can not only systematically reduce the number of senescent cells in multiple tissues of naturally aging mice, but also improve their muscle function and significantly reduce the level of SASP factor, thereby improving the aging-related phenotype of aged mice as a whole.

[0106] Repeat the above experimental method, using E9CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 20 CQNGGVCVSYKYFSRIRRCSCPRK can also systematically improve the aging pathological characteristics of naturally aging mice.

[0107] Example 7

[0108] E y -huPA 24 It targets and sensitizes human senescent cells, triggering NK cell killing.

[0109] To investigate the stimulatory effect of different amounts of glutamate on NK cells, five human senescent cell chimeric peptides were synthesized, with the sequence E. y LNGGTCVSNKYFSNIHWCNCPKKF, where y = 1, 2, 4, 8, 16.

[0110] Young and senescent IMR90 cells were mixed at 4000 / well with different concentrations of E. y -huPA 24 After incubation, the cells were co-cultured with NK92 cells. Results showed that NK92 cells eliminated the cytotoxicity of senescent cells over a relatively wide E50 range. y -huPA 24 Improvement was observed at certain drug concentrations, but not in young cells (Figures 11a-e). This indicates E y -huPA 24 It can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0111] E at a concentration of 5 μM y -huPA 24 After co-incubating with NK92 cells for 12 hours, the cell culture supernatant was collected, and the IFN-γ content secreted by NK cells was detected by ELISA. The results showed that E y -huPA 24 It can effectively stimulate NK cells to release IFN-γ (Figure 11f), and the effect is particularly significant when the number of glutamates is greater than 4.

[0112] E with a concentration of 5 μM coupled with 5-FAM y -huPA 24 After co-incubating with young and senescent IMR90 cells for 20 minutes, the interaction between young and senescent cells and E was detected by flow cytometry. y -huPA 24 The binding situation of E. The results show that E y -huPA 24 The binding to senescent cells is specific (Figure 11g-h), and E y -huPA 24 The targeting specificity to senescent cells is negatively correlated with the number of glutamate molecules.

[0113] In addition, the study investigated whether a functional unit can simultaneously possess two functions (targeting + immune sensitization).

[0114] Young or senescent cells were stained with the fluorescent dye CFSE and then inoculated with 0, 5, and 50 μM huPA. 24In the presence of [a specific ingredient], NK92 cells were co-incubated for 6 hours, followed by the addition of propidium iodide dye. The content of dead cells in young or senescent cells was detected by flow cytometry. The results (Figure 11i) showed that [the cell count in huPA] was [high / low]. 24 Under the influence of huPA, NK92 cells showed increased killing ability against senescent cells, and the killing effect of NK92 cells on senescent cells was significantly stronger than that on young cells. These results suggest that huPA... 24 It can simultaneously have both targeting and immune-sensitizing functions.

[0115] Example 8

[0116] E4-huPAz (4≤z≤24) targets and sensitizes human senescent cells, triggering NK cell killing.

[0117] In order to study huPA 24 Whether partial fragments in the sequence are sufficient to meet the targeting function, three senescent cell chimeric peptides (E4-huPA6, E4-huPA9, E4-huPA) were synthesized. 11 The sequences are E4SNKYFS, E4VSNKYFSNI, and E4VSNKYFSNIHW. The targeting regions of the three chimeric peptides consist of 6, 9, and 11 amino acid residues, respectively, all derived from huPA. 24 Part of the sequence. The activation function of the four glutamate repeat units for NK cells has been verified in Example 7.

[0118] Young and senescent IMR90 cells were incubated with different concentrations of E4-huPAz at 4000 cells / well and then co-cultured with NK92 cells. The results showed that the cytotoxicity of NK92 cells against senescent cells was improved at a wider range of E4-huPAz concentrations, but this effect was not observed in young cells (Figures 12a-c). This indicates that the three E4-huPAz formulations can effectively bind to NK cells and enhance the interaction between NK cells and senescent cells.

[0119] NK92 cells were co-incubated with 5 μM E4-huPAz for 12 hours, and the cell culture supernatant was collected. The IFN-γ content secreted by NK cells was detected by ELISA. The results showed that E4-huPAz could effectively stimulate NK cells to release IFN-γ (Figure 12d).

[0120] Young and senescent IMR90 cells were co-incubated with 5 μM 5-FAM-conjugated E4-huPAz for 20 minutes. Flow cytometry was used to detect the binding of 5-FAM-E4-huPAz to both young and senescent cells. The results showed that E4-huPAz specifically bound to senescent cells (Figure 12e-f). Furthermore, the targeting specificity of E4-huPAz to senescent cells was positively correlated with the length of the targeting sequence.

[0121] Example 9

[0122] To alleviate ovarian aging in both naturally aging mouse models and chemotherapy-induced aging mouse models.

[0123] (1)E16-uPA 24 Relieving ovarian aging in a mouse model induced by chemotherapy

[0124] Six- to eight-week-old female C57BL / 6 mice were weighed and randomly divided into two groups. The model group (n=6) received an intraperitoneal injection of cyclophosphamide (75 mg / kg), while the control group (n=3) received an equal volume of physiological saline solution. Seven days later, the model group mice were randomly divided into two subgroups (n=3 each) and injected intraperitoneally with E16-uPA. 24 (50 mg / kg) (modeling group) or an equal volume of physiological saline (modeling control group), injected every other day. Two weeks later, the estrous cycle of mice was detected by vaginal cell smears. Mice in the interestrous phase were weighed, and blood samples were collected after anesthesia. The ovaries of the mice were also removed and weighed. After the mouse blood samples were allowed to stand, they were centrifuged to collect serum. Blank wells, standard wells, and sample wells were set up in the enzyme-labeled plate of the ELISA kit. 50 μL of standard was added to each well. 40 μL of sample diluent was added to each sample well, followed by 10 μL of sample to be tested. After sealing the plate, the plate was placed in a 37°C incubator for 30 minutes. The concentrated wash buffer in the kit was diluted 20 times, the sealing film was removed, the liquid was discarded, and each well was filled with wash buffer. After standing for 30 seconds, the buffer was discarded. This process was repeated 5 times, and the plate was then dried. 50 μL of enzyme-labeled reagent was added to each well, except for the blank wells. The plate was then incubated again at 37°C for 30 minutes and washed. Add 50 μL each of chromogenic reagent A and B to each well, gently vortex to mix, and incubate at 37°C for 10 minutes in the dark. Stop the reaction by adding 50 μL of stop solution to each well. Measure the absorbance of each well on a microplate reader at 450 nm, using a blank well as the zeroing point. Mouse ovarian tissue was embedded in paraffin and stained with hematoxylin and eosin, and the number of antral follicles in the ovaries was counted.

[0125] The results showed that the levels of estradiol, follicle-stimulating hormone, and luteinizing hormone in mice were significantly different from those in the healthy group in the model control group, indicating that the chemotherapy-induced ovarian aging model was effective (Figure 13a). The treated model drug group showed significant differences from the model control group, approaching those of healthy mice, indicating that E16-uPA... 24 It has a significant therapeutic effect in a chemotherapy-induced ovarian aging model. Figure 13b shows that the ovarian weight ratio in the treatment group was significantly different from that in the untreated control group, and Figure 13c shows that the number of antral follicles in the ovaries was also greater after treatment, indicating that the ovaries in the treatment group were younger and healthier.

[0126] (2)E16-uPA 24 Ovarian aging in a mouse model that alleviates natural aging

[0127] Nine-month-old female C57 / BL6 mice under physiological conditions underwent ovarian senescence. E16-uPA was administered via intraperitoneal injection. 24 Administer 50 mg / kg or an equivalent volume of physiological saline every other day. Two weeks later, detect the estrous cycle of mice via vaginal cell smears. The specific method is as follows: Use a pipette to draw approximately 20 μL of physiological saline, gently insert it into the mouse's vaginal opening, slowly expel the saline, and then gently aspirate the liquid 2-3 times to rinse and collect exfoliated cells. Place the aspirated cell suspension onto a clean glass slide. After the droplet dries, add a drop of Giemsa working solution to the droplet and stain for 10 minutes. After staining, rinse away the excess stain very slowly with running water, and observe cell morphology under a microscope to determine the estrous cycle of the mouse.

[0128] Figures 14a and 14b show the tail vein injection of E16-uPA into mice every other day. 24 Intraperitoneal injection of E16-uPA 24 Estrogenic cycles in mice were measured 45 days after drug administration. The mice in Figure 14c were injected intraperitoneally with an equal volume of physiological saline. Compared to tail vein administration, intraperitoneal administration of E16-uPA... 24 Afterwards, the estrous cycles of two mice were essentially normalized (proestrus, estrus, metestrus, diaestrus, diaestrus, proestrus...). Compared with the saline group, both tail vein and intraperitoneal administration reduced the frequency of diaestrus, indicating that E16-uPA... 24 It has the effect of adjusting the stagnant state of mouse ovaries after natural aging to a younger, healthier, and more active state.

[0129] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were all found to effectively delay ovarian aging in mice.

[0130] Example 10

[0131] E16-uPA 24 It alleviates skin aging in a naturally aging mouse model.

[0132] Twenty-two-month-old C57BL / 6 mice were randomly divided into three groups and administered subcutaneous medication to a fixed area on the back of the mice every other day. The mice were injected with saline, 5 mg / kg of E16-uPA, or other appropriate medications. 24 and 15 mg / kg of E16-uPA 24 Three weeks later, skin tissue was taken from the intervention site for histopathological characterization and detection of aging-related proteins.

[0133] The results showed that subcutaneous administration of E16-uPA 24 It can significantly increase dermal-epidermal junction (DEJ) convolution, dermal thickness, and epidermal thickness, and stimulate collagen synthesis in aged mice (Fig. 15a, cf). Furthermore, the expression of aging-related proteins in the skin tissue at the intervention site is significantly downregulated, indicating that E16-uPA... 24 It has the effect of alleviating aging in mice and restoring the function of aging skin.

[0134] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK were also found to alleviate aging in mice and restore the function of aging skin.

[0135] Example 11

[0136] Treatment of a mouse model of macular degeneration.

[0137] Eight-week-old C57BL / 6 mice were randomly divided into two groups. Mice were induced to develop a skin model by a single tail vein injection of 35 mg / kg sodium iodate solution. Drug administration began two days after model initiation, with intervention administered via tail vein injection every other day. The mice were injected with either saline or 10 mg / kg E16-uPA. 24 HE staining results showed that sodium iodate induced decreased thickness of the extraretinal nuclear layer (ONL) and increased RPE layer thickness in mice due to the accumulation of RPE cells, which is consistent with the pathological characteristics of age-related dry macular degeneration. E16-uPA... 24 Drug intervention significantly improved ONL and RPE layer thickness in mice (Figures 16a-c). Retinal P16 immunofluorescence staining indicated that E16-uPA... 24 Treatment reduced the number of senescent cells with high P16 expression in the retinal region (Fig. 16d). Furthermore, senescence-related proteins P16, P53, and uPAR were all significantly downregulated after treatment (Fig. 16e), suggesting E16-uPAR... 24 It has a good therapeutic effect on age-related macular degeneration.

[0138] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK has also been found to have a good therapeutic effect on macular degeneration.

[0139] Example 12

[0140] It slows tumor progression and reduces chemotherapy toxicity.

[0141] Clinical chemotherapy for tumors induces senescence in both tumor cells and normal cells. Senescent tumor cells exhibit drug resistance and metastasis-promoting properties; senescence in normal cells disrupts tissue homeostasis and affects patient health. This process utilizes E16-uPA... 24 Eliminating senescent cells may enhance anti-cancer treatment and reduce the side effects of chemotherapy.

[0142] Female C57BL / 6N mice aged 8-12 weeks were subcutaneously injected with 5×10⁻⁵ mg / L on their backs. 6 Mouse lung cancer LLC cell lines expressing luciferase. Five days later, mice were divided into four groups for treatment. Treatment included PBS, BLM (10 mg / kg), and E16-uPA. 24 (50 mg / kg) or BLM (10 mg / kg) and E16-uPA 24 Mice were treated with a combination therapy of 50 mg / kg. Starting on day 6, for 6 weeks, mice received two intraperitoneal injections of BLM weekly to establish a tumor chemotherapy-induced aging model, while simultaneously receiving three tail vein injections of E16-uPA weekly. 24 .

[0143] Figure 17a shows that, although only E16-UPA 24 The therapeutic effect on tumor size was not significant, but compared with BLM alone, BLM and E16-UPA showed better therapeutic effects. 24 The combination therapy significantly enhanced tumor regression. Figures 17b and 17c show that the combination therapy significantly reduced the aging-related β-galactosidase staining areas in tumor tissue increased by BLM treatment, as well as the fibrotic areas in the lungs caused by BLM treatment. Figure 17d shows that the combination therapy prolonged the survival of tumor-bearing mice. Figure 17e shows that the number of CD161c-positive NK cells in mouse tumor tissue significantly increased after combination therapy treatment. These results suggest that E16-uPA... 24 It slows tumor progression and reduces chemotherapy toxicity.

[0144] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK was also found to slow tumor progression and reduce chemotherapy toxicity.

[0145] Example 13

[0146] Continuing the study of the effects of Example 6 on natural aging in mice

[0147] It improves the liver, kidney and lung function in naturally aging mice.

[0148] To further evaluate E16-uPA 24 To investigate its role in systemic anti-aging, we examined its effects on liver and kidney function in 22-month-old C57BL / 6J aged mice. The mice were divided into a control group and an E16-uPA group. 24 Treatment group (50 mg / kg, administered via tail vein 3 times per week for 8 consecutive weeks), positive control D+Q treatment group (dasatinib 50 mg / kg + quercetin 5 mg / kg, administered via tail vein 3 times per week for 8 consecutive weeks), and young mice were used as a healthy control group.

[0149] First, the expression levels of uPAR and p16 in liver and kidney tissues were detected by Western blotting. The results showed that the expression of uPAR and p16 was significantly increased in the elderly control group, while E16-uPAR expression was significantly lower. 24 Both D+Q treatment significantly reduced its expression (Figure 18a–f).

[0150] Subsequently, serum liver and kidney function indicators were measured. The results showed that E16-uPA... 24 It can reduce serum ALT, AST, and γ-GT levels in aged mice (Figure 18g–i), suggesting improved liver function; simultaneously, E16-uPA... 24 It also significantly reduced serum creatinine, uric acid, and blood urea nitrogen levels (Figure 18j–l), indicating improved renal function.

[0151] Further transcriptome heatmap analysis revealed that E16-uPA 24 The expression of multiple aging-related genes was significantly downregulated in the kidney (Fig. 18m), liver (Fig. 19a), and lung (Fig. 19b), indicating that it can improve aging-related transcriptional features at multiple tissue levels.

[0152] In summary, E16-uPA 24 It can not only reduce the expression of aging markers in the liver and kidneys of naturally aging mice, but also significantly improve their liver and kidney function indicators, and exert a systemic anti-aging effect by regulating the expression of aging-related genes in multiple tissues.

[0153] Repeat the above experimental method, using E8CQNGGVCVSYKYFSRIRRCSCPRK and E respectively. 24 CQNGGVCVSYKYFSRIRRCSCPRK has also been found to significantly improve liver and kidney function and achieve systemic anti-aging effects.

[0154] Example 14

[0155] Improves cardiac function in mice after cardiac fibrosis.

[0156] E1-uPA was evaluated using a mouse model of cardiac fibrosis.24 and E16-uPA 24 Effects on cardiac function. Mice were anesthetized with 4% pentobarbital and fixed on a temperature-controlled surgical board. They were intubated and connected to a ventilator (respiratory rate 75 breaths / min, tidal volume 5–7 mL). The skin of the left chest was incised, the pectoral muscles were dissected, and part of the ribs were removed along the 3rd–4th intercostal space to expose the heart. The pericardium was incised, and the left anterior descending coronary artery (LAD) was ligated with 8-0 polypropylene suture approximately 2 mm distal to the left atrial appendage. Paleness at the apex of the heart indicated successful modeling. The thoracic cavity was then sutured, and the animals were allowed to awaken.

[0157] The experiment was divided into four groups: sham surgery group (Sham), cardiac fibrosis group (MI), cardiac fibrosis + E1-uPA group, and cardiac fibrosis group (MI + E1-uPA). 24 Group(MI+E1-uPA 24 ) and cardiac fibrosis + E16-uPA 24 Group (MI+E16-UPA) 24 (n=4 in each group). The treatment group received E1-uPA via tail vein injection after modeling. 24 Or E16-uPA 24 (50 mg / kg), administered every other day.

[0158] Echocardiography was performed at baseline and on postoperative day 7 using a Vevo 2100 or Vevo F2 system under 2.5% isoflurane anesthesia to acquire parasternal long-axis and short-axis two-dimensional and M-mode images. Left ventricular function parameters were quantitatively analyzed using Vevo LAB software.

[0159] The results showed that, compared with the cardiac fibrosis group, E1-uPA 24 and E16-uPA 24 All treatments improved cardiac function. Specifically, EF and FS levels increased (Figure 20b, c), and the degree of dilation of LVIDs and LVIDd decreased (Figure 20d, e). E1-uPA... 24 With E16-uPA 24 There were no significant differences between them. The above results suggest that this type of peptide can improve cardiac contractile function to some extent after cardiac fibrosis.

[0160] Furthermore, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A molecule for targeting senescent cells to stimulate endogenous immunity, characterized in that, The molecule contains at least two basic units. One of the basic units is a unit with targeting functions for senescent cells, senescent tissues, or the senescent microenvironment. Another basic unit is the unit used to stimulate endogenous immunity; Or the molecule contains at least one basic unit. The basic unit is a unit that can simultaneously have the function of targeting senescent cells, senescent tissues or senescent microenvironments and stimulate endogenous immunity.

2. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 1, characterized in that, The unit having the function of targeting senescent cells, senescent tissues or senescent microenvironment is one or more of the following: antibody targeting specific antigens on the surface of senescent cells, ligand molecule targeting receptors highly expressed on senescent cells, polypeptide targeting senescent cells, nucleic acid aptamer targeting senescent cells, polysaccharide targeting senescent cells, and molecule targeting senescent microenvironment.

3. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 2, characterized in that, The antibody targeting specific antigens on the surface of senescent cells is an antibody against the urokinase-type plasminogen activator receptor.

4. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 2, characterized in that, The ligand molecule that targets the receptor highly expressed in senescent cells is a urokinase-type plasminogen activator.

5. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 2, characterized in that, The polypeptide targeting senescent cells is a urokinase-type plasminogen activator binding domain polypeptide.

6. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 2, characterized in that, The polysaccharide that targets senescent cells is β-galactose, which targets β-galactosidase, a marker of senescent cells.

7. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 1, characterized in that, The unit used to stimulate endogenous immunity is a molecule that can activate metabolic glutamate receptors or a molecule that can stimulate immune cells in aging tissues or the aging microenvironment.

8. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 7, characterized in that, The molecule that can activate metabolic glutamate receptors is one or more of a repeating unit of polyglutamate and a glutamate agonist. The immune cells are natural killer cells, macrophages, T cells, or dendritic cells.

9. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 5, characterized in that, The amino acid sequence of the urokinase-type plasminogen activator binding domain polypeptide molecule is all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

10. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 9, characterized in that, The amino acid sequence of the urokinase-type plasminogen activator binding domain polypeptide molecule is one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, and VSNKYFSNIHW.

11. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 9, characterized in that, The urokinase-type plasminogen activator binding domain polypeptide molecule is a linear molecule or its cyclic peptide form.

12. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 1, characterized in that, The molecule contains only one basic unit, which is a polypeptide molecule with an amino acid sequence of all or part of the sequence LNGGTCVSNKYFSNIHWCNCPKKF.

13. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 1, characterized in that, The amino acid sequence of the polypeptide molecule is one of LNGGTCVSNKYFSNIHWCNCPKKF, SNKYFS, VSNKYFSNI, and VSNKYFSNIHW.

14. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 8, characterized in that, The murine amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. x CQNGGVCVSYKYFSRIRRCSCPRK, where x is any integer.

15. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 9, characterized in that, The human amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. y LNGGTCVSNKYFSNIHWCNCPKKF is a sequence of all or part of the sequence, where y is any integer.

16. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 15, characterized in that, 1≤y≤24。 17. The molecule for targeting senescent cells to stimulate endogenous immunity as described in claim 15, characterized in that, The human amino acid sequence of the molecule used to target senescent cells and stimulate endogenous immunity is E. y LNGGTCVSNKYFSNIHWCNCPKKF, E y SNKYFS, E y VSNKYFSNI, E y One of the VSNKYFSNIHW.

18. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the molecule as described in any one of claims 1-17 for targeting senescent cells to stimulate endogenous immunity.

19. Use of the molecule for targeting senescent cells to stimulate endogenous immunity as described in any one of claims 1-17 in the preparation of a medicament for reversing age-related diseases or clearing senescent cells.

20. The use as described in claim 19, characterized in that, The relevant diseases are one of the following: organ fibrosis diseases such as liver fibrosis, kidney fibrosis, pulmonary fibrosis, and myocardial fibrosis; premature ovarian failure; macular degeneration; natural aging; synergistic tumor treatment; Alzheimer's disease; Parkinson's disease; atherosclerosis; cardiovascular dysfunction; loss of hematopoietic and skeletal muscle stem cell function; non-alcoholic fatty liver disease; chronic obstructive pulmonary disease; intestinal inflammation; diabetes; osteoarthritis; and osteoporosis.

21. A cosmetic composition, characterized in that, The cosmetic product contains the molecule as described in any one of claims 1-17 for targeting senescent cells to stimulate endogenous immunity.

22. A health product, characterized in that, The health product contains the molecule described in any one of claims 1-17 for targeting senescent cells to stimulate endogenous immunity.

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