A small-molecule chemical composition for partially chemically reprogramming and reversing body aging and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a small-molecule chemical composition for partially chemically reprogramming and reversing body aging and application. The small-molecule chemical composition for partially chemically reprogramming and reversing body aging comprises 0.1-0.7 mol parts of a WNT / beta-catenin agonist, 0.1-0.9 mol parts of a TGF-beta receptor inhibitor, 0.02-0.1 mol parts of a RAR agonist, 0.005-0.05 mol parts of a Smoothened receptor agonist, 0.02-0.1 mol parts of a VEGFR and PDGFR family multi-target inhibitor, 0.0002-0.002 mol parts of a histone methyltransferase inhibitor and 0.01-0.1 mol parts of a JAK1 / 2 inhibitor. The small-molecule chemical composition can improve the aging phenotype of tissues / organs, promote the recovery of body functions, reverse the aging process and promote physiological rejuvenation without inducing a pluripotent state.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a small molecule chemical composition for partially chemically reprogramming to reverse aging and its application. Background Technology

[0002] With rapid economic development and social progress, people's average lifespan has gradually increased, and population aging caused by aging has become one of the important social problems facing many countries around the world. Aging is a universal life phenomenon that affects multiple tissues and organs of the body. For example, the gradual decline in cell function leads to DNA damage, the decline in immune system function makes individuals more susceptible to disease, and the decline in physiological and cognitive functions leads to memory loss. The degeneration of various tissues and organs in an organism is a gradual process. In many animals, including vertebrates, the regeneration and repair capacity of vital organs is limited, especially with increasing age. In the later stages of aging, multiple organ failure syndromes such as cardiovascular and cerebrovascular diseases and cancer are often present. Compared to adult somatic cells, the tissues and organs of embryos or infants show a stronger ability to resist damage and stress and have higher regenerative vitality. Therefore, scientists have long been striving to find compositions and methods to restore tissues and organs from an aging state to a younger and more vibrant state of life, in order to delay and reverse aging and treat certain age-related injuries and diseases.

[0003] In recent years, with a deeper understanding of the mechanisms of aging, scientists have proposed the information theory of aging, pointing out that the aging process is driven by the gradual loss of youthful epigenetic information. Restoring this information through epigenetic reprogramming can catalyze age reversal and improve the function of damaged and aging tissues. This means that when tissues and organs age or are damaged, restoring the lost epigenetic information of cells can rejuvenate them; a typical example is epigenetic reprogramming technology. In 2006, four well-known "Yamano factors"—Oct4, Sox2, Klf4, and c-Myc (OSKM)—first reset the age of the aging epigenome, reprogramming mature somatic cells into younger induced pluripotent stem cells (iPSCs). However, this method also carries significant risks: resetting the epigenome may activate previously silenced oncogenes, leading to uncontrolled cell proliferation, and mature somatic cells, when transformed into iPSCs, may lose their original cellular identity. While partial reprogramming can be achieved by shortening the reprogramming time or removing c-Myc, thus reversing cellular aging while maintaining cellular identity, these methods still face potential obstacles to widespread use, including high costs and safety concerns related to introducing genetic material into the human body.

[0004] Compared to Yamanaka factor reprogramming, chemical reprogramming effectively regulates cell state and adjusts cell fate, resetting cells to pluripotency, subpotency, or performing partial reprogramming, without requiring the introduction of exogenous genes or complex experimental procedures. Chemical reprogramming typically involves epigenetic regulation, signaling pathway regulation, and cell cycle regulation, using specific small molecule compounds to modulate cell state. Studies have found that small chemical molecules such as sodium butyrate, α-ketoglutarate, valproic acid, and CHIR-99021 can restore cellular epigenetics, possessing the potential to revitalize cells and reverse transcriptome aging. However, aging is a complex phenomenon involving multiple physiological processes, and a single small molecule or a simple combination may not be sufficient to comprehensively address all types of aging. In existing technologies, the application of small chemical molecules and their compositions is limited to anti-aging effects at the cellular level or to the conversion of somatic cells into pluripotent or subpotent stem cells. Furthermore, there is a lack of data supporting their effectiveness and safety for anti-aging across the entire organism (CN202280018680.2, CN202410451919.6, CN201710026346.2). Deng Hongkui's team developed a small molecule combination capable of converting astrocytes into functional neurons in situ, but this application is also limited to local use. Therefore, finding a small molecule combination that can effectively reverse aging across the entire organism while avoiding side effects is a significant challenge.

[0005] The aging population has brought enormous pressure and challenges to the social security system, labor market, medical and health care, and family structure. We urgently need reprogramming small molecule chemical compositions and methods that can safely and effectively reverse aging, so as to restore aging cells or tissues and organs to a younger state, in order to reverse the aging of the entire organism. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a small molecule chemical composition for partially chemical reprogramming to reverse the aging of the body, the composition being able to reverse the aging of tissues / organs and restore their regenerative vitality.

[0007] To address the above problems, this invention discloses a small molecule chemical composition for partially chemically reprogramming to reverse aging, comprising the following components: 0.1–0.7 moles of a WNT / β-catenin agonist, 0.1–0.9 moles of a TGF-β receptor inhibitor, 0.02–0.1 moles of a RAR agonist, 0.005–0.05 moles of a smoothed receptor agonist, 0.02–0.1 moles of a VEGFR and PDGFR family multi-target inhibitor, 0.0002–0.002 moles of a histone methyltransferase inhibitor, and 0.01–0.1 moles of a JAK1 / 2 inhibitor.

[0008] Wherein, the WNT / β-catenin agonist includes at least one selected from MAY-262611, CHIR98014, Laduviglusib, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, SKL2001, or ARA014418; and,

[0009] The TGF-β receptor inhibitors include at least one of LY2109761, Pirfenidone, Repsox, SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, or SB505124; and,

[0010] The RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cisRA, BMS493, Adapalene, Vitamin A, or a Vitamin A derivative; and,

[0011] The smoothed receptor agonist includes at least one of SAG, SAG-d3, SAG hydrochloride, SAGdihydrochloride, or (Rac)-SAG; and,

[0012] The VEGFR and PDGFR family multi-target inhibitors include at least one of Linifanib, PP121, Vorolanib, Ki20227, SU11652, Sorafenib, Regorafenib, Sunitinib, Nintedanib, Ponatinib, Axitinib, Pazopanib, or SU 5402; and,

[0013] The histone methyltransferase inhibitors include at least one of EPZ004777, EPZ5676, GSK503, BIX01294, DZNep, DZNep·HCl, SGC0946, or Chaetocin; and,

[0014] The JAK1 / 2 inhibitors include at least one of Ruxolitinib, iJak-381, Tuspetinib, JAK1 / 2-IN-1, JAK-IN-1, Cerdulatinib, or JAK / HDAC-IN-2.

[0015] Optionally, the small molecule chemical composition for partially chemically reprogramming to reverse aging includes the following components: the WNT / β-catenin agonist Laduviglusib, the TGF-β receptor inhibitor RepSox, the RAR agonist TTNPB, the smoothed receptor agonist SAG, the VEGFR and PDGFR family multi-target inhibitor Linifanib, the histone methyltransferase inhibitor DZNep, and the JAK1 / 2 inhibitor Ruxolitinib.

[0016] WNT / β-catenin agonist 0.1–0.3 mol, TGF-β receptor inhibitor 0.3–0.6 mol, RAR agonist 0.02–0.05 mol, Smoothened receptor agonist 0.01–0.05 mol, VEGFR and PDGFR family multi-target inhibitors 0.01–0.05 mol, histone methyltransferase inhibitors 0.0002–0.0005 mol, and JAK1 / 2 inhibitors 0.03–0.05 mol.

[0017] Optionally, the small molecule chemical composition for partially chemically reprogramming to reverse aging includes the following components in molar amounts: 0.1–0.3 molar amounts of Laduviglusib, 0.3–0.6 molar amounts of RepSox, 0.02–0.05 molar amounts of TTNPB, 0.01–0.05 molar amounts of SAG, 0.01–0.05 molar amounts of Linifanib, 0.0002–0.0005 molar amounts of DZNep, and 0.03–0.05 molar amounts of Ruxolitinib.

[0018] Optionally, the small molecule chemical composition for partially chemically reprogramming to reverse aging comprises the following components in molar amounts: 0.2 molar amounts of Laduviglusib, 0.5 molar amounts of RepSox, 0.02 molar amounts of TTNPB, 0.01 molar amounts of SAG, 0.02 molar amounts of Linifanib, 0.0002 molar amounts of DZNep, and 0.04 molar amounts of Ruxolitinib;

[0019] Optionally, the small molecule chemical composition for partially chemically reprogramming to reverse aging comprises the following components in molar amounts: 0.7 molar amounts of Laduviglusib, 0.9 molar amounts of RepSox, 0.1 molar amounts of TTNPB, 0.05 molar amounts of SAG, 0.1 molar amounts of Linifanib, 0.002 molar amounts of DZNep, and 0.1 molar amounts of Ruxolitinib;

[0020] Optionally, the small molecule chemical composition for partially chemically reprogramming to reverse aging comprises the following components in molar amounts: 0.1 molar amount of CHIR-98014, 0.1 molar amount of SB431542, 0.02 molar amount of ATRA, 0.05 molar amount of SAG hydrochloride, 0.02 molar amount of Sorafenib, 0.0002 molar amount of EPZ004777, and 0.01 molar amount of JAK1 / 2-IN-1.

[0021] This invention also discloses a method of using a small molecule chemical composition for partially chemically reprogramming to reverse aging, comprising administering to a desired organism a small molecule chemical composition containing the following components: a WNT / β-catenin agonist, a TGF-β receptor inhibitor, a RAR agonist, a Smoothened receptor agonist, a VEGFR and PDGFR family multi-target inhibitor, a histone methyltransferase inhibitor, and a JAK1 / 2 inhibitor.

[0022] Specifically, the administration methods include intravenous injection, subcutaneous injection, intraperitoneal injection, airway administration, or spraying, with a dosage of 0.25–0.5 μL / g based on body weight.

[0023] The second technical problem to be solved by this invention is to provide the application of the above-mentioned small molecule chemical composition and the method for reversing aging in the field of anti-aging. This invention discloses the application of the small molecule chemical composition for partially chemically reprogramming to reverse aging and the method for using the small molecule chemical composition to reverse aging in the medical and health fields such as reversing aging and restoring the function of various tissues / organs.

[0024] The small molecule chemical composition for partial chemical reprogramming to reverse aging and / or the method for reversing aging using a small molecule chemical composition are used to achieve the use of a product having at least one of the following 1)-9):

[0025] 1) Reversing the aging phenotype of tissues / organs;

[0026] 2) Promotes tissue damage repair;

[0027] 3) Improve the body's behavioral and cognitive abilities;

[0028] 4) Improves the body's motor skills;

[0029] 5) Improves tissue / organ function;

[0030] 6) Promotes tissue / organ regeneration;

[0031] 7) Improve the expression of aging-related omics;

[0032] 8) Reversing the body's physiological age;

[0033] 9) Inhibit aging and age-related diseases.

[0034] The regulation may include mediating partial reprogramming of cells without eliminating cellular characteristics, reversing aging, reducing the appearance of aging, improving tissue function, improving organ function, promoting tissue repair, tissue survival, tissue regeneration, and tissue growth, inhibiting inflammatory responses, promoting angiogenesis, reducing scar formation, promoting organ repair, organ regeneration, and organ survival, downregulating the expression of aging-related genes and proteins, reversing the transcriptomic age of cells, tissues, and organs, promoting physiological rejuvenation, and inhibiting / delaying the progression of aging-related diseases.

[0035] Optionally, the tissues include epithelial tissue, muscle tissue, connective tissue, and nerve tissue; the organs include skin, lungs, heart, kidneys, spleen, liver, brain, testes, ovaries, etc.

[0036] The small-molecule chemical composition for partial chemical reprogramming to reverse aging described in this invention can effectively reverse the aging phenotype of tissues and organs, improve organ function, behavioral cognition and motor ability, regulate transcriptome gene expression, and regulate epigenetic expression profiles. Specifically, it includes: inhibiting the expression of aging-related genes, promoting the enrichment of functional genes related to skeletal muscle development and skin barrier establishment, enhancing the regenerative capacity of epidermal cells, reducing the outward manifestations of aging, reversing the decline of cardiac function, promoting blood circulation in tissues / organs, restoring muscle strength, improving behavioral cognition and motor ability, promoting the repair of tissue damage caused by inflammation, improving lung function in fibrotic lungs, reversing the biological age of organs including the heart, lungs, and brain, restoring aging or damaged bodies to a more youthful state, thereby inhibiting further aging and the progression of tissue diseases, increasing the regenerative and proliferative capacity of body tissues, effectively delaying the aging process, reversing aging, treating aging-related diseases, and resisting damage to tissues and organs caused by diseases.

[0037] The small-molecule chemical composition for partial chemical reprogramming to reverse aging described in this invention effectively solves the problems of weakened regenerative and repair capabilities and decreased resistance to disease in humans or animals as they age, and can effectively promote the recovery of tissue and organ functions in aging / damaged bodies.

[0038] Some existing technologies employ combinations of small chemical molecules that can reprogram somatic cells into pluripotent stem cells to prevent age-related histological changes and maintain youthful cells within tissues. In contrast, the small molecule combination in this invention can reverse the aging phenotype of tissues / organs without altering the original cellular identity, resulting in a more youthful state. Therefore, this invention represents a significant advancement over existing technologies in terms of improved safety, maintenance of original tissue structure and function, and direct reversal of existing aging markers.

[0039] The small-molecule chemical composition for partially reprogramming and reversing aging, the method for reversing aging using the small-molecule chemical composition, and the application described in this invention have the following technical advantages:

[0040] (1) Non-integration strategy: The technical solution disclosed in this invention adopts a small molecule chemical combination method to achieve a partial reprogramming strategy to reverse aging, rather than genetic engineering methods. It does not require the permanent integration of exogenous genes into the cell genome, thereby reducing the risk of gene mutation and carcinogenesis. Since there is no introduction of exogenous genes, it will not trigger an immune rejection response in the host, which is conducive to the long-term application of anti-aging therapy.

[0041] (2) High safety and controllability in vivo: The dosage of small molecule compounds is easy to control and can be precisely adjusted as needed to achieve the best reprogramming effect. This invention provides a safe and effective small molecule chemical combination that can reverse aging by analyzing the phenotypes and functions of multiple tissues / organs in the treated animals, animal behavior and cognition, expression of aging-related genes and prediction of physiological age.

[0042] (3) Easy to synthesize and scale up production: Small molecule compounds can be produced in large quantities through chemical synthesis, and the production cost is relatively low, which makes large-scale production and application possible and helps to reduce treatment costs.

[0043] (4) Great potential for clinical application: The small molecule chemical compositions for reversing aging through chemical reprogramming disclosed in this invention, and the methods for reversing aging using these small molecule chemical compositions, can provide new treatment strategies for various diseases caused by aging, such as degenerative diseases and decreased immune function. By restoring the vitality of tissues and organs through chemical reprogramming, it is expected to reverse aging and improve the overall health level of the body. Attached Figure Description

[0044] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0045] Figure 1The results of RT-PCR detection of genes Sox2 and P21 in renal progenitor cells 3 days after induction with the small molecule chemical composition in the comparative example and Example 1 of this invention (***P<0.001).

[0046] Figure 2 The results of skin phenotype tests of aged control and treatment group mice in Experiment Example 1 of this invention are as follows: a) Hair images and graying degree analysis results on days 5, 12 and 27 after drug administration; b) Skin epidermal HE staining images and thickness analysis results after 27 days of drug administration; c) Immunofluorescence staining images and fluorescence intensity analysis results of Ki67 expression after 27 days of drug administration; d) Observation and scoring of mouse body condition one month after drug administration (**P<0.01, ***P<0.001, ****P<0.0001).

[0047] Figure 3 The results of muscle phenotype tests of young control mice, aged control mice, and drug-treated mice one month after drug administration in Experiment Example 1 of this invention are as follows: a) Observation diagram of hind limb leg muscles and analysis results of forelimb muscle tension; b) HE staining diagram of hind limb leg muscle fibers and analysis results of muscle fiber cross-sectional area; c) CD31 immunofluorescence staining diagram of hind limb leg muscle tissue and analysis results of CD31 positive cells per unit area (*P<0.05, **P<0.01, ****P<0.0001).

[0048] Figure 4 The following are the respiratory tissue phenotypic test results of young control mice, aged control mice, and drug-treated mice one month after drug administration in Experiment Example 1 of this invention: a) HE staining images of alveolar tissue and analysis results of alveolar interstitial thickness; b) HE staining images of bronchial tissue and analysis results of bronchial wall thickness; c) Immunofluorescence staining images of CD31 expression and fluorescence coverage analysis results (*P<0.05, ****P<0.0001).

[0049] Figure 5 The results of the testicular tissue phenotypic test of young control mice, aged control mice, and drug-treated mice one month after drug administration in Experiment Example 1 of this invention are as follows: a) HE staining image of testicular tissue and analysis results of seminiferous tubule epithelial thickness; b) β-gal staining image and analysis results of positive area area; c) Immunofluorescence staining image of Ki67 expression and analysis results of positive cell proportion.

[0050] Figure 6 The results of small intestinal tissue phenotypic testing of young control mice, aged control mice, and drug-treated mice one month after drug administration in Experiment Example 1 of this invention are as follows: a) HE staining map of small intestinal tissue and analysis results of small intestinal villus length; b) β-gal staining map and analysis results of positive area area; c) Immunofluorescence staining map of Ki67 expression and analysis results of fluorescence intensity.

[0051] Figure 7 The results of behavioral tests on aged control mice and drug-treated mice in Experiment Example 2 of this invention are as follows: a) Open field route trajectory results and total distance analysis results before and after 13 days of drug treatment; b) Open field activity level heatmap and activity level analysis results before and after 13 days of drug treatment (*P<0.05, **P<0.01).

[0052] Figure 8 The following are the echocardiogram results and analysis results of young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 2 of this invention: a) Echocardiogram results; b) HE staining of tissues; c) β-gal staining; d) Analysis results of cardiac ejection fraction, shortening fraction, and area of ​​β-gal staining positive region (*P<0.05, **P<0.01, ****P<0.0001).

[0053] Figure 9 The images show the CD31 immunofluorescence staining patterns in the renal tubules (a) and glomeruli (b) of young control mice, aged control mice, and drug-treated mice one month after drug treatment in Example 2 of this invention, as well as the analysis results of CD31 positive cells per unit area (*P<0.05, **P<0.01, ****P<0.0001).

[0054] Figure 10 The following are the bulk transcriptome sequencing and bioinformatics analysis results of whole-body blood and brain tissues of young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 3 of this invention: a) Heatmap of age-characteristic gene expression in blood samples; b) Enrichment analysis of age-related gene sets in blood samples; c) Predictive analysis of brain transcriptome clock age.

[0055] Figure 11 The following are the bulk transcriptome sequencing and bioinformatics analysis results of muscle and skin tissues from young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 3 of this invention: a) Heatmap of age-characteristic gene expression and enrichment analysis of age-related gene sets in muscle tissue; b) Heatmap of age-characteristic gene expression in skin tissue; c) Enrichment analysis of age-related gene sets in skin tissue; d) Predictive analysis of skin transcriptome clock age.

[0056] Figure 12The following are the bulk transcriptome sequencing and bioinformatics analysis results of spleen tissue from young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 3 of this invention: a) Heatmap of age-characteristic gene expression in spleen tissue; b) GO gene functional enrichment map of differentially expressed genes in spleen tissue; c) Chronochron age prediction analysis map of spleen transcriptome.

[0057] Figure 13 The following are the bulk transcriptome sequencing and bioinformatics analysis results of lung tissues from young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 3 of this invention: a) Heatmap of age-characteristic gene expression in lung tissue; b) GO gene functional enrichment map of differentially expressed genes in lung tissue; c) Enrichment analysis map of age-related gene sets.

[0058] Figure 14 The following are the bulk transcriptome sequencing and bioinformatics analysis results of testicular tissue from young control mice, aged control mice, and drug-treated mice one month after drug treatment in Experiment Example 3 of this invention: a) Sperm characteristic gene expression heatmap; b) GO gene functional enrichment map and age-related gene set enrichment analysis map of differentially expressed genes in testicular tissue.

[0059] Figure 15 The following are the whole-genome bisulfite sequencing analysis results of heart, lung, and blood samples from young control mice, aged control mice, and drug-treated mice two months after drug treatment in Experiment Example 3 of this invention: a) epigenetic expression profiles; b) methylation age prediction analysis results; c) aging acceleration; d) KEGG pathway enrichment analysis results (*P<0.05, **P<0.01, ****P<0.0001).

[0060] Figure 16 The following are the results of lung and skin injury healing tests in healthy control mice, injury control mice, and injury treatment mice in Experiment Example 4 of this invention: a) Blood gas analysis results of lung function test; b) Images and analysis results of HE staining, Masson staining, and Ki67 immunofluorescence staining of lung tissue; c) Observation results of skin healing and wound area analysis (*P<0.05, **P<0.01, ***P<0.001).

[0061] Figure 17 The results of the analysis of one-minute walking distance and cognitive impairment rating in aged dogs during the baseline, treatment, and withdrawal periods in Example 5 of this invention are shown in (a), and the results of differential gene expression and functional enrichment analysis of bulk transcriptome before and after treatment are shown in (b) (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Detailed Implementation

[0062] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0063] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0064] Where specific experimental steps or conditions are not specified in the examples, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all raw materials used are commercially available products that are routinely obtainable by those skilled in the art.

[0065] Example 1

[0066] This embodiment provides a small molecule chemical composition for partially chemically reprogramming to reverse aging.

[0067] The small molecule chemical composition comprises the following molar amounts of small chemical molecules: 0.2 molar amounts of Laduviglusib, 0.5 molar amounts of RepSox, 0.02 molar amounts of TTNPB, 0.01 molar amounts of SAG, 0.02 molar amounts of Linifanib, 0.0002 molar amounts of DZNep, and 0.04 molar amounts of Ruxolitinib.

[0068] The small chemical molecules can be dissolved in DMSO or water to form storage solutions. To ensure the solubility and stability of the drugs in the mixture, Laduviglusib, RepSox, TTNPB, SAG, Linifanib, and Ruxolitinib are dissolved in DMSO, and DZNep is dissolved in water.

[0069] The storage solutions of the aforementioned small chemical molecules are mixed to form the small molecule chemical composition, and DMSO is used to make up to the required volume to obtain a composition solution. The composition solution comprises the following components at the following concentrations: 0.2 mM Laduviglusib, 0.5 mM RepSox, 0.02 mM TTNPB, 0.01 mM SAG, 0.02 mM Linifanib, 0.0002 mM DZNep, and 0.04 mM Ruxolitinib.

[0070] Example 2

[0071] This embodiment provides a drug for partially chemically reprogramming to reverse aging, which includes the small molecule chemical combination for partially chemically reprogramming to reverse aging as described in Example 1.

[0072] The drug formulation is obtained by diluting the composition solution in Example 1 with PBS buffer at a ratio of 20:1 to 50:1.

[0073] The methods of using some of the drugs mentioned above that chemically reprogrammable to reverse aging are as follows:

[0074] The administration of a certain amount of a preparation containing a small molecule chemical composition to the body, via methods such as intravenous injection, subcutaneous injection, intraperitoneal injection, airway administration, or nebulization.

[0075] Example 3

[0076] This embodiment provides a small molecule chemical composition for partially chemically reprogramming to reverse aging.

[0077] The small molecule chemical composition comprises the following components at the following concentrations: 0.7 moles of Laduviglusib, 0.9 moles of RepSox, 0.1 moles of TTNPB, 0.05 moles of SAG, 0.1 moles of Linifanib, 0.002 moles of DZNep, and 0.1 moles of Ruxolitinib.

[0078] The preparation process of the composition is as shown in Example 1.

[0079] Example 4

[0080] This embodiment provides a small molecule chemical composition for partially chemically reprogramming to reverse aging.

[0081] The small molecule chemical composition comprises the following components in concentrations: 0.1 moles of CHIR-98014, 0.1 moles of SB431542, 0.02 moles of ATRA, 0.05 moles of SAG hydrochloride, 0.02 moles of Sorafenib, 0.0002 moles of EPZ004777 and 0.01 moles of JAK1 / 2-IN-1.

[0082] The preparation process of the composition is the same as in Example 1.

[0083] Comparative Example

[0084] Compared to Example 1, this comparative example provides a series of small molecule chemical compositions that, compared to Example 1, lack Laduviglusib, RepSox, TTNPB, SAG, Linifanib, DZNep, and Ruxolitinib, respectively.

[0085] The different components of the aforementioned small molecule chemical compositions were diluted 1000:1 in renal progenitor cell culture medium and used for induction culture of renal progenitor cells. After 3 days of culture, cells were collected, and total RNA was extracted using the Trizol method. The RNA was reverse transcribed into cDNA using a reverse transcription kit (Novizan), following the kit's instructions. The cDNA was used as a template, and the template fragment was amplified using an RT-PCR kit. The expression of the reprogramming factor Sox2 and the aging-related gene P21 was detected using the GAPDH gene as an internal control. The results are shown in [Figure number missing]. Figure 1 .

[0086] Sox2 helps preserve the stem cell properties of cells, while high expression of P21 is generally considered a marker of aging in the body or specific tissues. Compared to the small molecule chemical composition (C7) provided in Example 1, the small molecule compound lacking any component—namely, Laduviglusib, RepSox, TTNPB, SAG, Linifanib, DZNep, and Ruxolitinib—showed relatively poor upregulation of the reprogrammed gene Sox2 and downregulation of the aging-related gene P21. This indicates that each component in Example 1 plays an indispensable role in the small molecule chemical composition, working together to achieve optimal effects, such as promoting partial cellular reprogramming or inhibiting the aging process. Therefore, the presence of all components is necessary to ensure effective reversal or delay of aging.

[0087] Experimental Example 1

[0088] This embodiment provides an application of a small-molecule chemical composition for partially chemically reprogramming to reverse aging and improve the phenotype of tissues / organs in aged organisms. The dosing groups described in the following experiments were administered via injection using the drug described in Example 1.

[0089] In this embodiment, 17-month-old C57BL / 6 aged mice were used as experimental subjects and randomly divided into an aged control group and an aged drug treatment group, with 2-month-old young C57BL / 6 mice serving as the young control group. During the experiment, the drugs were administered continuously for 4 weeks, with the aged mice receiving subcutaneous and intraperitoneal injections once a week. The aged drug treatment group received the composition solution described in Example 1 at a dosage of 0.25 μL / g per mouse based on its body weight. Before injection, the composition solution was diluted to 200 μL with PBS buffer per mouse. The aged control group received 200 μL of PBS buffer per mouse. Morphological and functional assessments were performed on the skin, muscle, and respiratory phenotypes of the aged mice.

[0090] Skin phenotype testing: On days 5, 12, and 27 after drug administration, mouse coat color was photographed and recorded. ImageJ software was then used to quantitatively analyze the degree of graying of the coat color using the same threshold. On day 27 after drug administration, a 3 cm long and 1 cm wide area under the left rib of the mouse was removed and paraffin-embedded. Paraffin tissue was sectioned at 5 μM, and sections were stained with hematoxylin and eosin (HE) and immunofluorescence to observe skin surface thickness and cell proliferation activity. Results and analysis of mouse coat color, HE staining of epidermal tissue, Ki67 staining, and other analytical results are shown below. Figure 2 .

[0091] Muscle phenotype testing: One month after drug administration, the skin of the right hind limb of mice in the three groups was removed, and muscle mass was observed and photographed. Forelimb muscle strength was tested using a mouse dynamometer. Simultaneously, hind limb leg muscle tissue was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) and CD31 immunofluorescence staining to assess muscle fiber size and muscle blood supply adequacy. Bright field images of the right hind limb muscle, forelimb strength test results, HE staining images of muscle fibers, fiber thickness analysis results, CD31 staining images, and analysis results are shown below. Figure 3 .

[0092] Respiratory tissue phenotypic testing: One month after drug administration, the left upper lobe of the mouse lung was selected for paraffin embedding. Paraffin tissue was sectioned at a thickness of 5 μM. The sections were stained with senescence-related β-galactosidase (β-gal), CD31 immunofluorescence staining, and HE staining to observe the expression of senescence markers in the perialveolar tissue, vascular distribution, alveolar interstitial thickness, and bronchial wall thickness. The results of β-gal staining, CD31 immunofluorescence staining, HE staining, and analysis are shown below. Figure 4 .

[0093] Phenotypic testing of testicular and small intestinal tissues: One month after drug administration, testicular and small intestinal tissues from mice were paraffin-embedded, sectioned, and then stained with hematoxylin and eosin (HE), Ki67 immunofluorescence staining, and β-gal staining. The staining and analysis results of testicular and small intestinal tissues are shown below. Figure 5 and Figure 6 .

[0094] like Figure 2 As shown in the figures, a) are the hair images and graying analysis results on days 5, 12, and 27 after drug administration; b) are the HE staining images and thickness analysis results of the skin epidermis of mice in the young control group, the aged control group, and the drug-treated group after 27 days of drug administration; c) are the immunofluorescence staining images and fluorescence intensity analysis results of Ki67 expression; d) are the overall appearance and body condition scores of mice in each group one month after drug administration (**P<0.01, ***P<0.001, ****P<0.0001). The results showed that compared with the aged control group, the graying of the coat color in the aged drug-treated group was significantly reduced, and the coat color remained basically stable after 12 days of drug administration, no longer turning further gray. Meanwhile, the epidermal cells of both the young control group and the aged drug-treated group were more tightly arranged, the epidermal thickness was significantly higher than that of the aged control group, and the Ki67 signal intensity in the skin tissue was higher. One month after treatment, the body condition scores of the aged mice in the drug-treated group were significantly better than those of the aged control group, and approached the normal levels of the young control group. Specifically, the aged mice in the drug-treated group had smoother, denser fur, reduced secretions, increased activity, and a generally good mental state. Graying fur and thinning back fur are natural signs of aging in C57BL / 6 mice. These data indicate that the small-molecule chemical composition effectively improved the overall health of aged mice and reversed their aging phenotypes. The small-molecule chemical composition can enhance the proliferative capacity of epidermal cells, giving them higher regenerative vitality, thereby effectively reversing the fur color of C57BL / 6 mice, inhibiting further graying, and increasing epidermal thickness to enhance the skin's resistance to external stimuli.

[0095] Figure 3This study included observations of hind limb leg muscles in young control mice, aged control mice, and the drug-treated mice one month after drug administration. Results included forelimb muscle tensile strength analysis, HE staining of hind limb leg muscle fibers, cross-sectional area analysis of muscle fibers, CD31 immunofluorescence staining of hind limb leg muscle tissue, and analysis of CD31-positive cells per unit area. Observations revealed that the leg muscles of young control mice and aged drug-treated mice were more abundant and rosy. The muscle strength of aged drug-treated mice was significantly enhanced compared to the aged control group, approaching the muscle strength level of young mice. Simultaneously, the thigh muscle fiber thickness and CD31-positive cell ratio of aged drug-treated mice were similar to those of young mice and significantly higher than those of the aged control group. These results indicate that the small-molecule chemical composition can effectively enhance blood supply and microcirculation in the muscles of aged mice, promote increased muscle fiber thickness, thereby improving muscle strength, preventing age-related muscle loss and strength decline, and rejuvenating leg muscles, providing a new possibility for delaying or reversing age-related muscle degeneration.

[0096] Figure 4 This study presents HE staining images of airway tissues, alveolar interstitial thickness, bronchial wall thickness analysis, β-gal staining images and analysis results, and immunofluorescence staining images and fluorescence coverage analysis results of CD31 expression in young control mice, aged control mice, and the drug-treated mice one month after drug treatment. Airway tissue staining and analysis results showed that, compared with young control mice, aged control mice exhibited significantly thickened alveolar interstitial tissue and bronchial walls, decreased CD31 expression, and increased β-gal expression. Furthermore, a large number of inflammatory cells were found infiltrating the thickened areas near the airways, suggesting that the mice may experience repeated inflammatory responses in the lungs with age, leading to abnormal proliferation of the alveolar interstitial tissue and bronchial walls. This abnormal thickening can lead to a decrease in vital capacity and respiratory mechanical elasticity, affecting lung respiratory function. After drug treatment, the alveolar interstitial tissue and bronchial walls of aged mice were significantly thinner, inflammatory cell infiltration was significantly reduced, CD31 expression was increased, β-gal expression was significantly decreased, and the alveolar interstitial thickness returned to a level not significantly different from that of young control mice. These results indicate that small molecule chemical compositions can effectively improve the inflammatory environment and aging phenotype in the lungs of aged mice, repair abnormal lung tissue proliferation caused by inflammation and aging, and provide a more suitable microenvironment for lung cell survival that is more similar to that of younger mice.

[0097] Figure 5This paper presents the histological analysis results of testicular tissue from young control mice, aged control mice, and aged mice treated with the drug one month after drug administration. The results showed that, compared with the aged control group, the seminiferous tubule epithelium thickness was significantly increased in the aged group treated with the drug, indicating that the small molecule chemical composition effectively promoted the structural and functional recovery of testicular tissue. Simultaneously, β-gal expression was significantly reduced in the testicular tissue of the aged group treated with the drug, further validating the drug's role in alleviating the aging phenotype of testicular tissue. Furthermore, the number of Ki67-positive cells was significantly increased in the aged group treated with the drug, suggesting that the drug may improve the regenerative capacity of testicular tissue by promoting cell proliferation. These results indicate that the small molecule chemical composition not only effectively improved the morphological characteristics of testicular tissue in aged mice but also significantly reversed the aging phenotype of testicular tissue by reducing the expression of aging markers and enhancing cell proliferation, bringing its function close to the normal level of young mice.

[0098] Figure 6 This study presents the histological analysis results of the small intestine tissues of young control mice, aged control mice, and aged mice treated with the drug one month after drug administration. The results showed that, compared with the aged control group, the villus length of the small intestine in the aged drug-treated group was significantly increased, indicating that the small molecule chemical composition effectively promoted the structural repair and functional recovery of the small intestinal mucosa. Simultaneously, β-gal expression in the small intestine tissue of the aged drug-treated group was significantly reduced, further validating the drug's role in alleviating the aging phenotype of small intestinal tissue. Furthermore, the number of Ki67-positive cells was significantly increased in the aged drug-treated group, suggesting that the drug may improve the regeneration and repair function of the small intestinal epithelium by enhancing cell proliferation. These results combined indicate that the small molecule chemical composition not only effectively improved the morphological characteristics of the small intestine tissue in aged mice but also significantly reversed the aging phenotype of the small intestine tissue by reducing the expression of aging markers and promoting cell proliferation, restoring its structure and function to near-normal levels found in young mice.

[0099] Therefore, the small molecule chemical composition and method for reversing aging by partial chemical reprogramming as defined in this invention can effectively reverse the aging phenotypes of tissues and organs such as skin, muscles, respiratory tract, small intestine, and testes by enhancing cell regeneration, improving blood circulation, and inhibiting the development of inflammation, thereby restoring the body to a youthful state and delaying the aging process.

[0100] Experimental Example 2

[0101] This embodiment provides an application of a small-molecule chemical composition that uses partial chemical reprogramming to reverse aging and improve behavioral cognition and organ function.

[0102] The preparation and use method of the small molecule chemical composition are as described in Examples 1 and 2.

[0103] In this embodiment, 17-month-old C57BL / 6 aged mice were used as experimental subjects and randomly divided into an aged control group and an aged drug-treated group, with 2-month-old young C57BL / 6 mice serving as the young control group. The aged mice were administered subcutaneous and intraperitoneal injections once a week. The aged drug-treated group received injections at a dosage of 0.25 μL / g per mouse based on their body weight. Before injection, the composition solution was diluted to 200 μL with PBS buffer per mouse. The aged control group received 200 μL of PBS buffer per mouse. Open field experiments were conducted on the aged mice before and on day 13 after drug administration. The open field measured 40 cm in length and 40 cm in width, and the experiments were conducted in a darkroom in a quiet environment. During the experiments, the mice ran freely in the open field for 5 minutes, and their activities were recorded in real-time via video recording. After recording, the Labmaze V3.0 maze video tracking system was used to analyze the recordings and assess indicators such as the mice's movement trajectory, distance and speed, and activity level to determine the impact of small molecule chemotherapy on the motor ability and behavior of aging mice. Results are available in the table below. Figure 7 One month after drug administration, a comprehensive cardiac function assessment was performed on the three groups of mice using echocardiography. The echocardiography results and related analyses are detailed below. Figure 8 Simultaneously, mouse kidney tissue was collected for CD31 immunofluorescence staining and observation in the renal tubules and glomeruli. The staining and analysis results are shown below. Figure 9 .

[0104] Figure 7 This paper presents the open field trajectory diagrams, total distance analysis, and open field activity heatmaps and analysis results for aged control mice and drug-treated mice after 13 days of treatment. Analysis of mouse movement trajectories revealed that the total distance traveled in the open field by aged control mice was significantly lower than before drug administration, while the total distance traveled in the open field by aged drug-treated mice significantly increased after 13 days of treatment, and their movement trajectories became more complex. Before drug administration, aged mice explored only parts of the open field, but after drug administration, their movement trajectories covered the entire open field area. These data indicate that the small molecule chemical composition can effectively improve the motor and spatial exploration abilities of mice. Analysis of mouse activity heatmaps showed that the activity level of aged drug-treated mice in the open field after drug administration was significantly higher than before drug administration and in the simultaneously treated aged control group. The red areas of high activity expression in aged drug-treated mice after drug administration covered all four corners of the open field, while the high activity expression values ​​in aged control mice were only covered in one corner. These data suggest that aged mice treated with the drug exhibited more active exploratory behavior, reflecting the positive effects of the small molecule chemical composition on the cognitive function or curiosity of aged mice.

[0105] Figure 8This study presents echocardiograms and analysis results of young control mice, aged control mice, and the drug-treated group one month after drug administration. Results showed significant improvements in several key cardiac function indicators in the aged drug-treated group, including cardiac pumping function and myocardial shortening fraction. Specifically, the cardiac pumping function of the aged drug-treated group approached the normal level of young mice, indicating that the small molecule chemical effectively promoted cardiac blood output efficiency. The recovery of myocardial shortening capacity further corroborates the significant effect of this chemical on improving the contractility of aging cardiac muscle. The overall cardiac structure remained unchanged before and after administration, indicating that the drug did not adversely affect cardiac structure. Furthermore, the reduced SA-β-gal expression in the hearts of aged drug-treated mice further validated the composition's effect on improving the aging phenotype of the heart. These results indicate that the small molecule chemical composition not only effectively reversed the decline in cardiac function in aged mice but also significantly improved their myocardial contractility and structural integrity, restoring the cardiac function of aged mice to a state close to that of young mice.

[0106] Figure 9 Immunofluorescence staining images of CD31 in renal tubules and glomeruli of young control mice, aged control mice, and the drug-treated group one month after drug treatment, and analysis results of CD31-positive cells per unit area. The results showed that, compared with the young control mice, the aged control mice had a significantly reduced number of CD31+ cells in the renal tubules and glomeruli. After drug treatment, the number of CD31+ cells recovered to a level comparable to that of age-appropriate mice. CD31 is a marker of vascular endothelial cells, and increased expression of CD31 predicts angiogenesis. With increasing age and aging, various renal functions progressively decline. The use of small molecule chemical compositions may improve renal function and reduce renal histological damage by improving the renal microvascular network and promoting angiogenesis in nephrons.

[0107] The above results indicate that the small molecule chemical composition and method for reversing aging by partial chemical reprogramming as defined in this invention can effectively improve the exploratory behavior and exercise level of aging organisms, reverse the decline of cardiac function, and improve the renal microvascular network, providing a possibility for improving the behavioral and cognitive abilities of the elderly population and restoring the health of organs such as the heart and kidneys.

[0108] Experimental Example 3

[0109] This embodiment provides an application of a small molecule chemical composition that uses partial chemical reprogramming to reverse aging, thereby reducing the expression of aging-related genes and reversing physiological age.

[0110] The preparation and use method of the small molecule chemical composition are as described in Examples 1 and 2.

[0111] In this embodiment, 17-month-old C57BL / 6 aged mice were used as experimental subjects and randomly divided into an aged control group and an aged drug-treated group, with 2-month-old young C57BL / 6 mice serving as the young control group. Drug administration was performed continuously for two months, with the aged mice receiving subcutaneous and intraperitoneal injections once a week. The aged drug-treated group received injections at a dosage of 0.25 μL / g per mouse based on body weight. Before injection, the drug solution was diluted to 200 μL with PBS buffer per mouse. The aged control group received 200 μL of PBS buffer per mouse. One month after drug administration, whole-body blood, brain, spleen, lung, muscle, skin, and testicular tissue samples were collected from the mice and sent to Novogene for bulk transcriptome sequencing. GSEA enrichment analysis of age-related genes was performed using the AgeAnno public database, and transcriptome clock age prediction analysis was performed using a pre-constructed cell age prediction model. Detailed analysis results are available in [link to analysis]. Figures 10 to 14 Two months after drug administration, whole-genome bisulfite sequencing was performed on blood, lung, and heart tissue samples from mice and sent to Novogene. Methylation age prediction analysis was conducted using the Steve Horvath 2022 epigenetic clock database, and related age accelerations were calculated. Detailed analysis results are available in [link to analysis]. Figure 15 .

[0112] Figure 10 Bulk transcriptome sequencing and bioinformatics analysis of whole-body blood and brain tissue from young control mice, aged control mice, and treated mice one month after drug administration. Results showed that in the treated aged mice, the expression of aging-related genes was downregulated, while the expression of youth-related genes was upregulated, resulting in a significantly different transcriptome expression profile compared to the aged control mice. Gene enrichment analysis further indicated that the blood gene expression profile of the treated mice was enriched with more youth-related genes, while the expression of aging-related genes was significantly reduced. Furthermore, age prediction analysis revealed a significant reversal of the biological age of the brains in these mice after treatment. These results suggest that small-molecule chemical composition therapy can not only significantly alter gene expression patterns in the blood, bringing them closer to a youthful state, but may also have positive effects on body organs such as the brain, bringing their function and structure closer to a youthful state.

[0113] Figure 11Bulk transcriptome sequencing and bioinformatics analysis of muscle and skin tissues from young control mice, aged control mice, and treated mice one month after drug administration. Results showed that the transcriptome expression profiles of muscle and skin in the aged treated mice differed significantly from those in the aged control group, but were more similar to those in the young control group, with high expression of many youth-related genes. Skin age prediction results indicated a significant reversal of biological age in the aged treated mice after drug administration. This demonstrates that small-molecule chemical compositions can effectively reverse the aging process of skin and muscle and promote a more youthful state.

[0114] Figure 12 Bulk transcriptome sequencing and bioinformatics analysis of spleen tissues from young control mice, aged control mice, and treated mice one month after drug administration. Results showed that the spleen transcriptome expression profile of treated aged mice was significantly different from that of untreated aged control mice. The spleens of treated mice exhibited lower levels of aging-related gene expression and higher expression of youth-related genes, making their transcriptome expression profiles more similar to those of young control mice. Differential gene enrichment analysis indicated that the expression of genes related to cell development was significantly upregulated in the spleens of treated mice, while the expression of genes related to inflammation was downregulated. Based on age prediction results, treatment with the small molecule chemical composition can effectively reverse the aging process of the spleen, making its functional characteristics tend towards a more youthful state.

[0115] Figure 13 Bulk transcriptome sequencing and bioinformatics analysis of lung tissues from young control mice, aged control mice, and treated mice one month after drug administration. Results showed that the lung transcriptome expression profiles of treated aged mice were more similar to those of young control mice, but significantly different from those of untreated aged control mice. Differential gene enrichment analysis revealed that the expression of genes related to oxygen transport was significantly upregulated in the lungs of treated mice, while the expression of genes related to inflammation was significantly downregulated. Furthermore, enrichment analysis of aging-related genes showed a significant reduction in their expression in the lungs of treated mice. These results collectively indicate that treatment with small-molecule chemical compositions helps restore the youthful characteristics of the lungs and improve their physiological function.

[0116] Figure 14Bulk transcriptome sequencing and bioinformatics analysis of testicular tissues from young control mice, aged control mice, and drug-treated mice one month after drug treatment. Results showed that in the treated aged drug-treated mice, the testicular transcriptome expression profile revealed a significant increase in the expression of markers for elongated spermatocyte subtypes and mature spermatocytes. Differential gene enrichment analysis further indicated that genes related to spermatogenesis were significantly upregulated in the drug-treated mice. Combined with the results of age-related gene set enrichment analysis, these findings suggest that the small-molecule chemical composition can effectively reverse the physiological age of the testes and enhance their spermatogenic function.

[0117] Figure 15 This report presents whole-genome bisulfite sequencing analysis of heart, lung, and blood samples from young control mice, aged control mice, and treated mice two months after drug administration. Results showed that the epigenetic expression profiles in the heart, lungs, and blood of aged mice under treatment were significantly altered. Furthermore, age prediction based on methylation revealed a significant decrease in the epigenetic age of the treated mice, with a negative aging acceleration, demonstrating that drug treatment effectively reversed the epigenetic age of mice. KEGG pathway enrichment analysis showed high enrichment of longevity-related pathways in the treated mice, while pathways related to aging were highly enriched in the aged control mice, indicating that aging in multiple tissues and organs was reversed after drug treatment.

[0118] The above results indicate that the small molecule chemical composition and method for reversing aging by partial chemical reprogramming as defined in this invention can reduce the expression of aging-related genes in the whole body blood and multiple organs. By enriching youth-related genes and enhancing their expression, it can play a broad anti-aging role, thereby reversing the physiological age of the body.

[0119] Test Example 4

[0120] This embodiment provides an application of a small-molecule chemical composition that uses partial chemical reprogramming to reverse aging and promote tissue repair. The preparation and use of the small-molecule chemical composition are as described in Examples 1 and 2.

[0121] In this embodiment, 2-month-old C57BL / 6 mice were used as experimental subjects and randomly divided into three groups: a healthy control group, an injury control group, and an injury treatment group. Mice in both the injury control and injury treatment groups were given bleomycin via airway inhalation one week prior to drug administration to establish a pulmonary fibrosis model. After successful establishment of the pulmonary fibrosis model, drug administration was initiated to the injury control and treatment groups. The administration frequency was twice weekly, once subcutaneously and once intraperitoneally, for a total administration period of 28 days. The injury treatment group received an injection at a dosage of 0.25 μL / g per mouse based on their body weight. Before injection, the composition solution was diluted to 200 μL with PBS buffer per mouse. The injury control group received 200 μL of PBS buffer per mouse. One week after drug administration, mechanical skin injury was performed on all three groups of mice. A 1 cm diameter circular incision was cut into the back of each mouse using a sterilized instrument. The area of ​​skin injury on the mouse's back was then continuously statistically quantified. Mice were treated with a small molecule chemical composition for 28 days, and blood gas analysis was performed. Lung tissue was collected for paraffin embedding, HE staining, Masson staining, and Ki67 immunofluorescence staining. Results of lung and skin lesion healing in mice are shown below. Figure 16 .

[0122] Figure 16 The results of blood gas analysis, HE staining, Masson staining, and Ki67 immunofluorescence staining of lung tissue were compared between healthy control mice, injury control mice, and treatment mice. The results showed that in the injury control group, after bleomycin injury, the partial pressure of oxygen decreased significantly, the partial pressure of carbon dioxide increased significantly, and significant bullae appeared in the alveolar region of the mice, accompanied by a large amount of inflammatory cell infiltration, severe collagen fiber deposition, and low expression of the proliferation marker Ki67. Skin wound healing was also slow. In contrast, the partial pressures of oxygen and carbon dioxide in the treatment group recovered to levels close to those of healthy mice. Compared to the injury control group, the treatment group showed significantly reduced inflammatory cell infiltration in the lung tissue, milder collagen fiber deposition, significantly increased expression of the proliferation marker Ki67, and skin wound healing speed comparable to that of the healthy control group. These results indicate that the small molecule chemical composition can effectively reduce bleomycin-induced inflammatory cell infiltration and alveolar damage in the lungs, inhibit the progression of pulmonary fibrosis, and promote the proliferation of autologous cells to help repair damaged areas, thereby restoring lung respiratory function and skin injury healing speed in mice to levels close to those of healthy mice.

[0123] Therefore, the small-molecule chemical compositions and methods for reversing aging through partial chemical reprogramming as defined in this invention may accelerate the healing of bodily injuries through multiple pathways, such as enhancing cell proliferation and differentiation, regulating inflammatory responses, and promoting blood oxygenation. This discovery provides strong experimental evidence for the potential application of small-molecule chemical compositions in the treatment of bodily trauma or injury.

[0124] Experimental Example 5

[0125] This embodiment provides an application of a small-molecule chemical composition for reversing aging in aged dogs using partial chemical reprogramming. The preparation and application methods of the small-molecule chemical composition are as described in Examples 1 and 2.

[0126] In this embodiment, a 12-year-old male dog was used as the experimental subject. The experiment was designed in three phases: a four-week baseline period before drug administration, during which the basic condition of the dog was recorded; a three-week treatment period, during which the dog was administered the drug via intravenous infusion once a week, with the dosage injected at 0.5 μL / g based on the dog's body weight. Each intravenous infusion was pre-mixed with PBS to a final volume of 50 ml. After drug administration was discontinued, the dog was observed for three weeks as a withdrawal period. During these three phases, the dog underwent a one-minute walking distance test and cognitive impairment assessment weekly. The one-minute walking distance test was conducted under leash guidance; the dog explored its activity area independently within one minute, and the total distance traveled was measured and calculated. Simultaneously, blood samples were taken from the dogs before and after drug administration for bulk transcriptome sequencing, followed by differential gene expression and functional enrichment analysis. The relevant detection and analysis results are detailed below. Figure 17 .

[0127] Figure 17 This study analyzed the one-minute walking distance and cognitive impairment rating of aged dogs at baseline, during treatment, and after treatment, as well as the differential gene expression and functional enrichment analysis of the transcriptome before and after treatment. Results showed that compared to baseline, the walking distance of aged dogs significantly increased and the cognitive impairment score significantly decreased during the treatment period. Three weeks after treatment, the treated dogs maintained a high walking distance and a low cognitive impairment score. Furthermore, after drug administration, several genes related to skeletal muscle development and skin barrier establishment were significantly enriched in the aged dogs, consistent with observed increases in motor function and decreased dandruff. Enrichment of genes related to cardiovascular system development, reproductive system development, wound healing, and angiogenesis was also observed. This indicates that small-molecule chemical compositions can effectively improve the bodily functions of aged dogs.

[0128] The experiments described in Examples 1 to 5 were performed on Examples 3 and 4 respectively, and the experimental results obtained were basically consistent with those of Examples 1 to 5.

[0129] Therefore, the small-molecule chemical compositions and methods for reversing aging through partial chemical reprogramming as defined in this invention can alter the expression patterns of functional genes in aging organisms, improve bodily functions, and alleviate cognitive impairment and motor abilities. These findings provide strong support for the potential application of small-molecule chemistry in delaying age-related functional decline. Obviously, the above embodiments are merely illustrative examples and not intended to limit the implementation. Those skilled in the art can make various variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. A small-molecule chemical composition for partially chemically reprogramming to reverse aging, characterized in that, include: WNT / β-catenin agonist 0.1–0.7 mol, TGF-β receptor inhibitor 0.1–0.9 mol, RAR agonist 0.02–0.1 mol, Smoothened receptor agonist 0.005–0.05 mol, VEGFR and PDGFR family multi-target inhibitor 0.02–0.1 mol, histone methyltransferase inhibitor 0.0002–0.002 mol, and JAK1 / 2 inhibitor 0.01–0.1 mol; The WNT / β-catenin agonist includes at least one of MAY-262611, CHIR98014, Laduviglusib, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, SKL2001, or ARA014418; The TGF-β receptor inhibitors include at least one of LY2109761, Pirfenidone, Repsox, SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, or SB505124; The RAR agonist includes at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cisRA, BMS493, Adapalene, Vitamin A, or a Vitamin A derivative. The smoothed receptor agonist includes at least one of SAG, SAG-d3, SAG hydrochloride, SAGdihydrochloride or (Rac)-SAG; The VEGFR and PDGFR family of multi-target inhibitors include at least one of Linifanib, PP121, Vorolanib, Ki20227, SU11652, Sorafenib, Regorafenib, Sunitinib, Nintedanib, Ponatinib, Axitinib, Pazopanib, or SU 5402. The histone methyltransferase inhibitors include at least one of EPZ004777, EPZ5676, GSK503, BIX01294, DZNep, DZNep·HCL, SGC0946, or Chaetocin. The JAK1 / 2 inhibitors include at least one of Ruxolitinib, iJak-381, Tuspetinib, JAK1 / 2-IN-1, JAK-IN-1, Cerdulatinib, or JAK / HDAC-IN-2.

2. The small molecule chemical composition for partially chemically reprogramming to reverse aging according to claim 1, characterized in that, It comprises the following components in molar amounts: WNT / β-catenin agonist 0.1–0.3 molars, TGF-β receptor inhibitor 0.3–0.6 molars, RAR agonist 0.02–0.05 molars, Smoothened receptor agonist 0.01–0.05 molars, VEGFR and PDGFR family multi-target inhibitors 0.02–0.05 molars, histone methyltransferase inhibitors 0.0002–0.0005 molars, and JAK1 / 2 inhibitors 0.03–0.05 molars.

3. The small molecule chemical composition for partially chemically reprogramming to reverse aging according to claim 1, characterized in that, It includes the following components in molar amounts: 0.1–0.3 molars of Laduviglusib, 0.3–0.6 molars of RepSox, 0.02–0.05 molars of TTNPB, 0.01–0.05 molars of SAG, 0.02–0.05 molars of Linifanib, 0.0002–0.0005 molars of DZNep, and 0.03–0.05 molars of Ruxolitinib.

4. The small molecule chemical composition for partially chemically reprogramming to reverse aging according to claim 1, characterized in that, The small molecule chemical composition for partial chemical reprogramming to reverse aging comprises the following components in molar amounts: 0.2 molar amounts of Laduviglusib, 0.5 molar amounts of RepSox, 0.02 molar amounts of TTNPB, 0.01 molar amounts of SAG, 0.02 molar amounts of Linifanib, 0.0002 molar amounts of DZNep, and 0.04 molar amounts of Ruxolitinib.

5. The small molecule chemical composition for partially chemically reprogramming to reverse aging according to claim 1, characterized in that, The small molecule chemical composition for partial chemical reprogramming to reverse aging comprises the following components in molar amounts: 0.7 molar amounts of Laduviglusib, 0.9 molar amounts of RepSox, 0.1 molar amounts of TTNPB, 0.05 molar amounts of SAG, 0.1 molar amounts of Linifanib, 0.002 molar amounts of DZNep, and 0.1 molar amounts of Ruxolitinib.

6. The small molecule chemical composition for partially chemically reprogramming to reverse aging according to claim 1, characterized in that, The small molecule chemical composition for partial chemical reprogramming to reverse aging comprises the following components in molar amounts: 0.1 molar amount of CHIR-98014, 0.1 molar amount of SB431542, 0.02 molar amount of ATRA, 0.05 molar amount of SAG hydrochloride, 0.02 molar amount of Sorafenib, 0.0002 molar amount of EPZ004777, and 0.01 molar amount of JAK1 / 2-IN-1.

7. Use of the small molecule chemical composition for partially chemically reprogramming to reverse aging according to any one of claims 1-6 in the preparation of a product having at least one of the following effects (1) to (9): 1) Reversing the aging phenotype of tissues / organs; 2) Promotes tissue damage repair; 3) Improve the body's cognitive abilities in behavior; 4) Improves the body's motor skills; 5) Improves tissue / organ function; 6) Promotes tissue / organ regeneration; 7) Improve the expression of aging-related omics; 8) Reversing the body's physiological age; 9) Inhibit aging and age-related diseases.

8. A drug for partially chemically reprogramming to reverse aging, characterized in that, The drug for partially chemically reprogramming to reverse aging comprises the small molecule chemical composition for partially chemically reprogramming to reverse aging as described in any one of claims 1-5.

9. The medicament according to claim 8, characterized in that, The drug is an injectable or sprayable formulation.

10. The medicament according to claim 8, characterized in that, The drug also contains a pharmaceutically acceptable solvent; the weight ratio of the pharmaceutically acceptable solvent to the small molecule chemical composition is 20:1-50:1; The solvents acceptable for the drug are at least one of PBS, physiological saline, or compound electrolyte injection.

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