CN117205193A

CN117205193A - Application of Salvianolic Acid A (SAA) as novel anti-aging medicine raw material in cell aging, tumor treatment and longevity hit prolonging

Info

Publication number: CN117205193A
Application number: CN202211176616.5A
Authority: CN
Inventors: 孙宇
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2022-09-26
Filing date: 2022-09-26
Publication date: 2023-12-12
Also published as: WO2024067604A1

Abstract

The invention provides application of salvianolic acid A as a novel anti-aging drug raw material in cell aging, tumor treatment and longevity hit prolonging. The invention discovers that Salvianolic Acid A (SAA) can specifically target aging cells and has the effects of inhibiting the expression of aging-related secretion phenotype (SASP), removing the aging cells and recovering the proliferation activity of the aging cells. In addition, the anti-tumor agent can remove aged stromal cells in tumor microrings after treatment of genotoxic chemotherapeutic agents and reverse SASP expression trend, so that drug resistance of tumors to the chemotherapeutic agents is reduced, and actual anti-tumor effects of the chemotherapeutic agents are remarkably promoted. Meanwhile, SAA can also obviously prolong the survival time of experimental animals in the later stage.

Description

Application of Salvianolic Acid A (SAA) as novel anti-aging medicine raw material in cell aging, tumor treatment and longevity hit prolonging

Technical Field

The invention relates to the field of biological medicine. In particular to application of Salvianolic Acid A (SAA) as a novel anti-aging drug raw material in cell aging, tumor treatment and longevity hit prolonging.

Background

Cell aging refers to a relatively stable and generally irreversible state of cell cycle arrest in eukaryotic cells, in which proliferating cells are tolerant to growth-promoting stimuli, usually caused by stress signals such as DNA damage. Replicative senescence means that normal cells stop dividing continuously after about 30-50 divisions (i.e. "Hayflick limit"). Replicative senescence is essentially induced by progressive shortening of telomeres. In each round of DNA replication, the telomeres gradually shorten, eventually reaching a critical length, preventing further replication, and stopping cell division. Shorter uncapped telomeres can elicit a DNA damage response, directly triggering senescence.

It is now widely accepted that, except for cell types with stem cell-like properties, only transformed malignant cells will replicate indefinitely, whereas non-transformed cells will not. Senescent cells are different from resting cells and terminally differentiated cells, in that resting cells are able to reenter the cell cycle. Senescent cells are characterized by morphological abnormalities, altered metabolic activity, chromatin remodeling, altered gene expression, increased lipofuscin, pronounced granularity, severe vacuolation, and the appearance of a pro-inflammatory phenotype known as the senescence-associated secretory phenotype (SASP). Disruption of nuclear membrane integrity was observed due to loss of nuclear lamin B1 expression. Senescent cells accumulate dysfunctional mitochondria and exhibit elevated levels of Reactive Oxygen Species (ROS). Increased lysosomal inclusion and altered lysosomal activity was also observed, which was manifested as an increase in the positive staining rate of beta-galactosidase at pH 6.0, making it a widely used marker of cellular senescence. The biological effects of aging are complex, and both protective and deleterious effects of aging cells have been described, depending primarily on the pathophysiological environment. For example, although aging may evolve as a mechanism to avoid malignant transformation of damaged cells, the occurrence of aging may lead to a number of age-related pathologies, including a range of clinical problems such as cancer, cardiovascular and cerebrovascular diseases, osteoporosis, arthritis, metabolic diseases, neurodegenerative symptoms, and the like.

Cell senescence is manifested by nuclear membrane invagination, chromatin contraction, increased cell volume, activation of multiple signaling pathways downstream including p53, p16.sup.INK4A/Rb, PI3K/Akt, foxO transcription factors, and mitochondrial SIRT 1. In addition to entering permanent proliferation arrest, senescent cells are often associated with a number of pathological features, including localized inflammation. Cell senescence occurs in damaged cells and prevents their proliferation in vivo. Cell damage can lead to obvious signs of cell aging under the influence of various external stimuli and internal factors. When the lesion accumulation reaches a certain limit, various macroscopic tissue degenerative changes and physiological aging phenotypes are presented in the tissue.

Of particular note, the expression level of inflammatory cytokines in senescent cells is significantly elevated, a phenomenon known as the senescent-associated secretory phenotype (SASP). The concept of SASP was first proposed by Coppe et al in 2008. They found that senescent cells promote the cancerous or malignant elevation of neighboring precancerous cells by secreting extracellular matrix proteins, inflammation-related factors, and cancer cell growth factors, and these proteins are referred to as SASP factors.

Senescent cells are involved in various physiological and pathological processes in the body mainly through 3 pathways: (1) Progressive accumulation of senescent cell gene expression and morphology changes can affect the function of the corresponding tissue; (2) Senescent cells limit the regenerative potential of stem cells and undifferentiated progenitor cells, resulting in a decrease in cell regeneration capacity; (3) Aging cells not only exhibit growth cycle arrest, but also release a large number of cytokines, chemokines, growth factors, proteases, etc. through the autocrine and paracrine pathways, affecting the microenvironment of neighboring cells and tissues, leading to and accelerating aging and related diseases, and a great deal of research in recent years has shown that SASP plays a central pathological role in this process. In addition, these factors secreted by senescent cells affect surrounding normal cells, while inhibition of SASP can delay body aging. Typical SASP factors include tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1a (IL-1 a), matrix Metalloproteinase (MMP), granulocyte-macrophage colony stimulating factor (GM-CSF), and plasminogen activator inhibitor-1 (PAI 1), etc., which promote the activation of the immune system, leading to the elimination of abnormal factors such as senescent cells in the tissue microenvironment by the body, exerting tumor-inhibiting functions. However, quite paradoxically, SASP may promote tumor progression by promoting angiogenesis by factors that are specific for secreted factors (e.g., VEGF, ANGPTL 4), extracellular matrix remodeling, or epithelial-to-mesenchymal transition (EMT). In addition, chronic inflammation induced by aging can cause systemic immunosuppression, and the chronic inflammation can also promote the occurrence and development of various aging-related diseases such as aging-related tissue injury and degeneration, organ dysfunction, cancer and the like.

Stimulation of DNA damage, telomere dysfunction, oncogene activation, oxidative stress, etc. can induce the appearance of SASP in cells, the mechanism of which is closely related to transcriptional cascades, autocrine loops, and sustained DNA damage responses. However, overexpression or inhibition of the senescent classical pathways p53 and p16.sup.INK4A/Rb did not affect SASP expression, indicating that although cycle arrest of senescent cells and SASP frequently co-occur, the regulatory pathways of the two do not overlap exactly. It has been reported that DNA damage response increases secretion of SASP factors IL-6 and IL-8 by activating the ataxia telangiectasia mutated gene, nemerheng fracture syndrome protein 1 and checkpoint kinase 2. The DNA Damage Response (DDR) is activated immediately after cell damage, and aging cells take about 1 week or more to develop mature SASP, and transient DNA damage responses do not induce cellular aging nor SASP, suggesting that there are other mechanisms in addition to DNA damage responses that co-induce SASP.

Epigenetic studies have been carried out in recent years in the field of SASP research. Sirtuins is a metabolic-related, NADH-dependent deacetylase and SIRT1 has been found to have life-prolonging effects in various models. SIRT1 in senescent cells inhibits the expression of SASP factors by deacetylating the IL-6 and IL-8 promoter regions histones H3K9 and H4K16, which are higher in acetylation than in control cells during cell senescence after SIRT1 knockout. microRNAs are a highly conserved single-stranded non-coding RNA of about 20-26 nucleotides in length that regulates gene expression in eukaryotic cells. Research results show that miR-146, miR-34, miR-21, miR-183 and the like can regulate aging cell SASP and can effectively inhibit excessive production of inflammatory cytokines. miR-146a/b can reduce the production of IL-1 receptor related kinase in human umbilical vein endothelial cells; conversely, inhibiting miR-146a/B can increase the activity of IL-1 receptor-related kinase, activate transcription factor NF- κB, and induce IL-6 and IL-8 to produce.

Epigenetic changes affect senescence by affecting DNA damage repair, telomere length, and metabolic pathways, or activating expression of senescence-associated genes and miRNAs. Various evidences indicate that changes in chromatin state are closely related to the control of cellular senescence. The cell may feel different senescence stimuli which activate signaling pathways driving changes in chromatin state. However, the way in which the senescence signal causes this change remains largely unknown. Therefore, the epigenetic aspect reveals the regulatory mechanism of cell senescence and the occurrence and development of specific phenotypes, and further reveals the key molecules with targeting value and the signal paths thereof, which are an emerging direction of the biological and senile medicine of the future, and the related exploration needs to be developed deeply, so that important scientific basis and potential intervention measures are provided for clinical medicine.

With the increasing aging of the global population, the interest in "active health, delaying aging" is growing, mainly based on a series of scientific evidence that the basic mechanisms of targeted aging can delay the occurrence or exacerbation of various aging-related chronic or non-infectious diseases. Thus, cellular aging has gained much attention as a potential target for preventing or treating a variety of aging-related diseases and improving health life.

The medicine for delaying senescence is mainly used for selectively clearing senescence cells by temporarily blocking survival pathways (senescent cell anti-apoptosis pathways SCAPs) which can protect the senescent cells from being regulated by apoptosis induction signals in the environment. Preclinical studies have shown that a class of drugs, senolytics, is expected to be applied in the future to delay, prevent or treat a variety of aging-related diseases.

While more and more experiments support that targeted cell senescence can treat a variety of senescence-associated diseases simultaneously, there are still stringent human clinical trials to help people better assess the benefits and risks of anti-senescence drugs. Although a number of internationally known inhibitors of SASP can significantly attenuate SASP, they do not substantially kill senescent cells. To pharmacologically reduce the burden on senescent cells, scientists are developing small molecules, polypeptides and antibodies of the nature "senolytics" (senescent cell clearing drugs) to selectively clear senescent cells. Researchers have made considerable progress in identifying other small molecule senolytic drugs and their actions since the discovery of senolytic drugs in 2015. The first article of research to find senolytic drugs is based on the hypothesis that senescent cells resist apoptosis, although senescent cells produce pro-apoptotic SASP factors to trigger self-death.

Indeed, studies have shown that pro-apoptotic pathways are indeed upregulated in senescent cells. Thus, aging cells rely on aging-related anti-apoptotic pathways (SCAPs) to mitigate SASP's' damage to themselves, a hypothesis that has been validated. The SCAPs were identified by bioinformatics methods (based on the expression profile of radiation-induced senescent human preadipocytes). Studies have been conducted to find that senescent cells have a dependency on SCAPs by in vitro RNA interference experiments and to identify SCAPs as a fatal weakness of senescent cells. This research finding ultimately contributed to the discovery of potential senolytic targets in the SCAP network as well as the discovery of the first senolytic drug, which included the combination (d+q) of dasatinib (a FDA approved tyrosine kinase inhibitor) and quercetin (quercetin), a flavanol found in many fruits and vegetables. In addition, there have been studies to identify a protein of the BCL-2 family that is resistant to apoptosis (BCL-XL) as a SASP component. Following this discovery, a third senolytic drug, navitocrax, was also identified, which is a BCL-2 family inhibitor. Researchers have now identified an increasing number of senolytics, including other synthetic small molecules, compounds extracted from natural products, and inhibitors targeting known SCAPs peptides. In addition, SCAPs have also been attracting attention as potential senolytic targets.

SCAPs required for senescent cell survival vary between cell types. For example, the SCAPs required for the survival of senescent human primary adipocytes are different from those in senescent human embryonic vein endothelial cells (HUVECs). This difference means that drugs targeting a single SCAP may not eliminate multiple aging cell types. Moreover, a number of studies have shown that most senolytics are indeed effective only on limited senescent cell types. For example, navitocrax is capable of targeting HUVECs, but is not effective on senescent human adipose progenitor cells. There is evidence that the efficacy of senolytics may vary even within a particular type of cell. For example, in human lung fibroblasts, navitocrax can target and kill senescent cells in an IMR-90 lung fibroblast-like cell line adapted to culture, but has little effect on senescent primary human lung fibroblasts. Thus, to determine the broad spectrum of actions of senolytics, extensive testing for a range of cell types is still required.

Under certain conditions, the frequency of use of senolytic drugs may depend on the rate of accumulation of senescent cells, which may vary depending on the environment in which cell senescence occurs. For example, repeated exposure to cancer therapies that destroy DNA or a sustained high fat diet may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics may reduce the risk of adverse reactions in the patient and allow for use of senolytics during health. Furthermore, intermittent administration can also reduce side effects caused by senolytics and reduce the likelihood of resistance in patients. In contrast to the case of anticancer drugs or antibiotics, the body cannot rely on cell proliferation to develop senolytics resistance because senolytics do not divide, and thus cannot obtain favorable mutations, which creates a good basis for the widespread use of senolytics in future clinics.

Disclosure of Invention

The invention aims to provide application of Salvianolic Acid A (SAA) as a novel anti-aging drug raw material in cell aging, tumor treatment and longevity hit prolonging.

The invention provides a novel anti-aging medicine raw material which plays an important role in cell aging, tumor treatment and longevity hit.

In a first aspect of the present invention there is provided the use of salvianolic acid a for the preparation of a formulation or pharmaceutical composition for:

a) Inhibiting senescence-associated secretion phenotype (SASP) expression;

b) Anti-aging or anti-aging cells;

c) Reduce the drug resistance of the tumor to the chemotherapeutic drugs; and/or

D) Prolonging the service life or the late-year survival time.

In another preferred embodiment, the senescence-associated secretion phenotype is a senescence-associated secretion phenotype caused by DNA damage; preferably DNA damage caused by chemotherapeutic agents or ionizing radiation.

In another preferred embodiment, the concentration of salvianolic acid A in the preparation or pharmaceutical composition for inhibiting SASP expression is 10-200. Mu.M, preferably 20-100. Mu.M, more preferably 40-60. Mu.M (e.g., 40, 45, 50, 55 or 60. Mu.M).

In another preferred embodiment, said anti-aging comprises inducing the ability of senescent cells to resume growth and/or proliferation.

In another preferred embodiment, said clearing senescent cells comprises inducing the senescent cells to enter a death program.

In another preferred embodiment, the concentration of salvianolic acid A in the preparation or pharmaceutical composition for eliminating senescent cells is > 100. Mu.M, preferably 200-3000. Mu.M, more preferably 200-2000. Mu.M (e.g., 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000. Mu.M).

In another preferred embodiment, the salvianolic acid A reduces the resistance of the tumor to the chemotherapeutic agent by inducing the entry of senescent cells in the tumor microenvironment into the death program, preferably by caspase-3/7 mediated induction of senescent cells in the tumor microenvironment into the death program.

In another preferred embodiment, the chemotherapeutic agent is a genotoxic agent.

In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: mitoxantrone, bleomycin, doxorubicin, or a combination thereof.

In another preferred embodiment, the tumor is selected from the group consisting of: prostate cancer, lung cancer, stomach cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof.

In another preferred embodiment, the salvianolic acid A does not affect or substantially does not affect proliferating cells.

In a second aspect of the invention, there is provided a pharmaceutical composition or combination comprising a therapeutically effective amount of:

c1 Salvianolic acid a, or a pharmaceutically acceptable salt thereof, as a first active ingredient;

c2 A chemotherapeutic agent as a second active ingredient,

wherein the chemotherapeutic agent is capable of inducing the appearance of senescent cells in the tumor microenvironment.

In another preferred embodiment, the chemotherapeutic agent is capable of inducing SASP expression in the tumor microenvironment; or can induce senescence marker p16 ^INK4A Up-regulating.

In another preferred example, the chemotherapeutic is mitoxantrone and the weight ratio of mitoxantrone to salvianolic acid A is 1:10-100; preferably 1:25-75; more preferably 1:40-60 (e.g., 1:45, 1:50 or 1:55, more preferably 1:50).

In another preferred example, the chemotherapeutic agent is bleomycin and the concentration of bleomycin is 1-100 μg/ml; preferably 20-70. Mu.g/ml; more preferably 40-60. Mu.g/ml (e.g. 45, 50 or 55. Mu.g/ml, more preferably 50. Mu.g/ml).

In another preferred example, the chemotherapeutic drug is doxorubicin, and the weight ratio of doxorubicin to salvianolic acid A is 1:20-200; preferably 1:50-150; more preferably 1:80-120.

In another preferred embodiment, the concentration of salvianolic acid A in the pharmaceutical composition is 10-200. Mu.M, preferably 20-100. Mu.M, more preferably 40-60. Mu.M (e.g., 40, 45, 50, 55 or 60. Mu.M), wherein salvianolic acid A is used to inhibit SASP expression.

In another preferred embodiment, the concentration of salvianolic acid A in the pharmaceutical composition is > 100. Mu.M, preferably 200-3000. Mu.M, more preferably 200-2000. Mu.M (e.g., 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000. Mu.M), wherein salvianolic acid A is used to induce aging cells into a death program.

In a third aspect of the present invention, there is provided a method for screening a substance capable of synergistically anti-tumor with salvianolic acid A, comprising the steps of:

(S1) providing a tumor tissue, treating the tumor tissue with a candidate substance;

(S2) detecting the aging condition of cells in the treated tumor tissue, thereby screening the substances which can cooperate with the salvianolic acid A to resist tumors.

In another preferred embodiment, the anti-tumor comprises: promoting tumor regression preferably includes clearing senescent cells in the tumor microenvironment.

In another preferred embodiment, said detecting a cell senescence condition comprises:

(i) Detecting the number of senescent cells;

(ii) Detecting SASP factor expression;

(iii) Detection of senescence marker p16 ^INK4A 。

In another preferred embodiment, if it is detected that the candidate agent treatment increases (e.g., increases by 10%, 20%, 30%, 40%, 50% or more) the number of senescent cells, SASP factor expression, and/or the senescence marker p16 in the tumor tissue ^INK4A The substance is considered to be a substance which can cooperate with salvianolic acid A to resist tumors.

In another preferred embodiment, the SASP factor includes IL6, CXCL8, SPINK1, WNT16B, GM-CSF, MMP3, IL 1A, but is not limited thereto.

In another preferred embodiment, the tumor tissue comprises tumor cells and stromal cells.

In another preferred embodiment, the detecting the cell senescence is detecting the senescence of stromal cells.

In another preferred embodiment, the method is in vivo screening or in vitro screening.

In another preferred embodiment, the anti-tumor effect comprises: tumor cell killing rate, tumor tissue volume reduction rate, tumor-bearing animal survival rate, but is not limited thereto.

In another preferred embodiment, the method further comprises the steps of: a control group is provided, which may be a no treatment control group.

In another preferred example, the substance includes a small molecule compound, a biological macromolecule, but is not limited thereto.

In another preferred embodiment, the candidate substance is a chemotherapeutic agent.

In another preferred embodiment, the method is a non-diagnostic non-therapeutic method.

In a fourth aspect of the invention, there is provided a method of non-therapeutically inhibiting cellular SASP expression, eliminating senescent cells, and/or reducing resistance of tumor cells to a chemotherapeutic agent in vitro comprising the steps of:

contacting salvianolic acid a with said cells, thereby inhibiting its expression of SASP, clearing said senescent cells, and/or reducing resistance of said tumor cells to a chemotherapeutic agent.

In a fifth aspect of the invention, there is provided a method of inhibiting SASP expression, anti-aging or eliminating aging cells, reducing tumor resistance to a chemotherapeutic agent, or extending life or late-year survival in a subject in need thereof, comprising the steps of:

Salvianolic acid a is administered to a subject in need thereof to inhibit SASP expression, to combat aging or eliminate aging cells, to reduce tumor resistance to chemotherapeutic agents, or to extend life or late survival.

In a sixth aspect of the invention, there is provided a method of treating a tumor in a subject in need thereof, comprising the steps of:

administering to a subject in need thereof a therapeutically effective amount of: chemotherapeutic agents, and salvianolic acid a.

In another preferred embodiment, the chemotherapeutic agent is a genotoxic agent, preferably the chemotherapeutic agent is capable of increasing SASP expression in a tumor microenvironment.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 shows the results of expanded human stromal cells PSC27 (early algebra such as p 10-20) after 7-10 days after staining by SA- β -Gal in vitro with the chemotherapeutic drug Bleomycin (BLER) at a concentration of 50 μg/ml. Upper, representative, lower, statistical data. CTRL, control cells; BLEO, bleomycin-treated cells. * P <0.001.

FIG. 2 shows the results of PSC27 cells after treatment with the chemotherapeutic drug Bleomycin (BLER) and after staining with BrdU. Upper, representative, lower, statistical data. CTRL, control cells; BLEO, bleomycin-treated cells. * P <0.0001.

Fig. 3 shows the results after PSC27 cells were treated with the chemotherapeutic drug Bleomycin (BLEO) and immunofluorescent stained with γh2ax (immunofluorescence staining). CTRL, control cells; BLEO, bleomycin-treated cells. * P <0.001. The fluorescent spots in the nucleus are classified into 4 types including individual cells of 0foci,1 to 3foci,4 to 10foci and >10foci, according to the number of fluorescent spots in the nucleus.

Figure 4 shows a flow chart of experiments for large-scale screening of libraries of organic chemicals to obtain small molecule compounds (including natural and synthetic) with anti-aging activity.

FIG. 5 shows a schematic chemical molecular structure of SAA.

FIG. 6 shows that Heatmap after RNA-seq data analysis shows up-regulation of a number of factor expression in senescent cells caused by BLER damage, but that there is not a few significant reversals after SAA treatment. Red star marks, typical SASP exocrine factors.

Figure 7 shows GSEA analysis results showing that the expression of SASP-specific molecular markers or related factors is centrally up-regulated in senescent cells caused by BLEO, but significantly reduced after SAA treatment of senescent cells.

Figure 8 shows GSEA analysis results showing that NF- κb molecular marker or related factor expression is centrally up-regulated in senescent cells caused by BLEO, but significantly reduced after SAA treatment of senescent cells.

Figure 9 shows a representative pathway on biological process for 100 molecules that KEGG pathway analysis SAA caused significant downregulation in senescent cells.

Figure 10 shows a representative pathway on cellular component for 100 molecules that KEGG pathway analysis SAA caused significant downregulation in senescent cells.

FIG. 11 shows the relative expression levels of a typical set of SASP molecules under conditions of treatment with varying concentrations of SAA in senescent cells induced by BLER. All data are normalized results compared to CTRL groups. * P <0.05; * P <0.01; * P <0.001.

FIG. 12 shows the determination of the aging of PSC27 by SA-. Beta. -Gal staining under conditions of increasing SAA concentration. P >0.05; * P <0.01; * P <0.0001. Wherein the P-values of SAA at concentrations of 100 μm,200 μm,400 μm,800 μm,1600 μm,2000 μm and 3000 μm are statistically significant for the cell positive ratios of these experimental groups compared to the data at 0 μm.

FIG. 13 shows representative pictures of PSC27 under various conditions after SA- β -Gal staining. Each group was repeated 3 times, arranged up and down. Scale, 30 μm.

FIG. 14 shows the survival of CCK 8-detected proliferating cells with senescent group cells at increasing SAA concentrations. The P-value at each SAA concentration is a significant difference between CTRL and BLEO groups after comparison. * P <0.01; * P <0.001; * P <0.0001.

Fig. 15 shows population doubling (population doubling, PD) testing of PSC 27. Cells were subjected to BLER damage at passage 10 (p 10), followed by SAA addition to the medium at day 8. The effect of SAA on the proliferation potential of cells was determined by comparative analysis of the multiplication values (PD) of CTRL, blo, SAA and BLEO/SAA groups. P >0.05; * P <0.001.

FIG. 16 shows the induction of caspase 3/7 activity during SAA treatment of senescent cells. PSC27 cells were gradually brought into senescence phase after being treated with BLER under culture conditions for 12 h. 200. Mu.M SAA was added to the medium of senescent cells starting at day 7, nucLight Rapid Red reagent was used to label the cells, and caspase 3/7 reagent (IncuCyte) was used for apoptosis detection. Caspase 3/7 activity was assayed every 4 hours (n=3).

FIG. 17 shows that Pan-caspase inhibitors (20. Mu.M QVD-OPh) reverse the senolytic activity of SAA (200. Mu.M SAA was used in this experiment, while 1.25. Mu.M ABT263 served as positive control; the latter was a recently reported senescent apoptosis inducer). Statistical differences were obtained by two-way ANOVA (Turkey' test).

FIG. 18 shows flow cytometry to determine apoptosis of PSC27 under several conditions. Q2, distribution region of early apoptotic cells; q3, distribution region of late apoptotic cells.

Figure 19 shows the survival and number of apoptosis after blo and/or SAA treatment of comparative analysis cells. * P <0.001; * P <0.0001.

Figure 20 shows a schematic representation of the mode of administration in mice in a preclinical trial. Human stromal cells PSC27 and cancer cells PC3 were transplanted into mice subcutaneously after in vitro mixing (1:4) to form transplantable tumors. After a plurality of treatment cycles under single drug or combined drug administration conditions, the mice are finally sacrificed and the change of the expression of molecules related to tumor tissues is pathologically analyzed.

Figure 21 shows that CTRL and BLEO-injured groups of PSC27 cells were transplanted into subcutaneous tissue of mice to form a graft tumor after in vitro mixing with PC3, or PC3 cells alone. Tumors were dissected and obtained at the end of week 8, and tumor volumes were detected and compared under each set of conditions. * P <0.01; * P <0.001; p <0.0001.

Figure 22 shows a schematic representation of the timing and mode of administration of pre-clinical trial mice. Once every two weeks, MIT (mitoxantrone) was administered intraperitoneally to mice on the first day of weeks 3/5/7, respectively. Mice were given intraperitoneal SAA, once a week, starting on day 5. After the end of the 8-week course, the mice were dissected and subjected to pathology identification and expression analysis.

Figure 23 shows a terminal volume statistical analysis of tumors. The chemotherapeutic agent MIT alone or in combination with the anti-aging agent SAA was administered to mice and the tumor sizes of each group were analyzed in comparison after the end of week 8.

FIG. 24 shows a comparison of cell senescence in lesions of PC3/PSC27 tumor-bearing animals in preclinical experiments. Representative pictures after SA- β -Gal staining. Scale, 100 μm.

FIG. 25 shows a parallel analysis of the percentage of SA- β -Gal staining positive cells in tumor tissue in mice. P >0.05; * P <0.05; * P <0.0001.

FIG. 26 shows the analysis of SASP-typical factor expression in epithelial and stromal cells in mouse foci by fluorescent quantitative PCR (qRT-PCR) assay. Stromal cells and cancer cells were specifically isolated by LCM techniques, total RNAs were prepared and used for SASP expression detection, respectively. P >0.05; * P <0.05; p <0.01.

FIG. 27 shows the status of matrix cell SASP factor expression in mouse foci after administration of veicle, MIT and MIT/SAA by fluorescent quantitative PCR (qRT-PCR) assay. * P <0.05; * P <0.01; p <0.001.

Figure 28 shows DNA damage and apoptosis ratios in groups of mice analyzed after specific isolation of cancer cells in lesions using LCM technology. P >0.05; * P <0.05; * P <0.01.

Figure 29 shows a picture analysis after immunohistochemical staining (immunohistochemical staining). The signals of Caspase 3 clear (CCL 3) in the lesions of the mice in each group are in sharp contrast. Scale, 200 μm.

FIG. 30 shows a comparison of Kaplan Meier data for disease-free survival of NOD/SCID mice after various dosing treatments. Animals of the Vehicle, MIT, SAA and MIT/SAA groups had tumor volumes exceeding 2000mm in vivo ³ When serious diseases are considered to occur, mice need to be sacrificed in time and their tumor-bearing conditions detected. P>0.05；**,P<0.01。

Figure 31 shows comparative analysis of mice body weight data at the end of the course of treatment under various dosing conditions. And P >0.05.

Figure 32 shows comparative analysis of the serological data of mice at the end of the course of treatment under the different dosing treatment conditions above. Creatinine, urine (kidney index), ALP and ALT (liver index) data were compared in parallel. And P >0.05.

Figure 33 shows comparative analysis of body weight data from immunized intact mice (C57 BL/6J) at the end of the course of treatment under various dosing conditions. And P >0.05.

Figure 34 shows a comparative analysis of the blood cell count of mice at the end of the course of treatment at different dosing treatment conditions in the pre-clinic. WBC, lymphocyte and neutrophil are compared in parallel per unit volume. And P >0.05.

Figure 35 shows a terminal volume statistical analysis of tumors. The chemotherapeutic agent DOX alone or in combination with the anti-aging agent SAA was administered to mice and the tumor sizes of each group were analyzed in comparison after the end of week 8.

Figure 36 shows a terminal volume statistical analysis of tumors. The chemotherapeutic agent DOC alone or in combination with the anti-aging agent SAA was administered to mice and the tumor sizes of each group were analyzed in comparison after the end of week 8.

Figure 37 shows a terminal volume statistical analysis of tumors. Chemotherapeutic agent VIN alone or in combination with anti-aging agent SAA was administered to mice and tumor sizes of each group were analyzed in comparison after the end of week 8.

Fig. 38 shows post-treatment survival curves of pre-clinical stage mice. Starting from 24 to 27 months of age, C57BL/6 mice underwent once every two weeks Vehicle or SAA intraperitoneal administration (Vehicle group n=80; SAA group n=91). Median survival (median survival) for each group of animals was calculated and indicated. P <0.0001.

Fig. 39 shows the overall (lifetime, or full length) survival curve of pre-clinical stage mice. Starting from 24 to 27 months of age, C57BL/6 mice underwent once every two weeks Vehicle or SAA intraperitoneal administration (Vehicle group n=80; SAA group n=91). Median survival (median survival) for each group of animals was calculated and indicated. * P <0.0001.

Detailed Description

Through extensive and intensive studies, the inventors of the present invention have developed for the first time the use of Salvianolic Acid A (SAA) as a novel anti-aging drug substance in cell aging, tumor treatment and longevity-prolonging therapies. In the invention, experiments show that Salvianolic Acid A (SAA) can specifically target aging cells, and has the effects of inhibiting the expression of aging-related secretion phenotype (SASP), removing the aging cells and recovering the proliferation activity of the aging cells. In addition, the anti-tumor agent can remove aged stromal cells in tumor microrings after treatment of genotoxic chemotherapeutic agents and reverse SASP expression, so that the drug resistance of tumors to the chemotherapeutic agents is reduced, and the anti-tumor effect of the chemotherapeutic agents is obviously promoted. Meanwhile, SAA can also obviously prolong the late survival time of the aged animals. On this basis, the present invention has been completed.

Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

As used herein, the term "pharmaceutically acceptable carrier" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount that produces a function or activity in and is acceptable to a human and/or animal. It will be appreciated by those of ordinary skill in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug being used, the severity of the disease, and the combination with other drugs.

Salvianolic Acid A (SAA)

Salvianolic acid A (Salvianolic acid A, SAA) is prepared by extracting root of Salvia Miltiorrhiza (Salvia miltiorrhiza Bge.) of Labiatae, and refining to obtain pale yellow crystal. Molecular formula C ₂₆ H ₂₂ O ₁₀ Dissolving in ethanol and diethyl ether. Melting point 315-323 ℃.

The SAA effects found in the present invention include:

1. inhibition of SASP expression: in senescent cells highly expressing SASP, SASP expression was significantly down-regulated after SAA treatment. In certain embodiments, this effect was demonstrated to be optimal at SAA concentrations of about 50. Mu.M.

2. Anti-aging or anti-aging cells: SAA is capable of promoting entry of senescent cells into the death program by inducing apoptosis. In certain embodiments, the effect is demonstrated to be concentration dependent under in vitro conditions, exhibiting a significant effect at SAA concentrations of about > 200 μM, and reaching a threshold when SAA concentrations are about 2000 μM, can reduce the percentage of surviving senescent cells to about 10%. The examples also demonstrate that SAA does not substantially affect proliferating cells at 3000. Mu.M, and has excellent specificity.

In addition, SAA can significantly enhance the population doubling capacity (PD) of stromal cells which are growth-arrested after genotoxic treatment (e.g., BLER treatment), enhance the proliferation capacity of the stromal cells, have no influence on the PD of the proliferated cells, and exhibit good selectivity.

3. Reduce the drug resistance of the tumor to the chemotherapeutic drugs: the present invention has been found by research that administration of certain chemotherapeutic agents (e.g., MIT) can induce the appearance of a large number of senescent cells in tumor tissue, and the up-regulation of SASP factor expression, which occurs primarily in stromal cells, such an increase in SASP factor will promote therapeutic resistance of peripheral cancer cells. While SAA is able to reverse this change to a large extent. Animal experiments also demonstrate that for transplanted tumors composed of PC3 cancer cells and primary PSC27 stromal cells, SAA alone has no significant effect on tumor volume, but unexpectedly can significantly reduce tumor volume in mice after MIT treatment by 55.1% compared to MIT. Survival experiments showed that SAA alone had no significant prolongation effect on survival of tumor-bearing mice, but unexpectedly, mice treated with the MIT/SAA combination had a prolongation of survival of at least 48.1% compared to the group treated with MIT alone. Meanwhile, urea, creatinine, liver enzymes or body weight of experimental mice do not have significant fluctuation, the integrity of an immune system and tissue homeostasis of key organs are not disturbed, and it is proved that an anti-aging agent combined with a conventional chemotherapeutic drug is likely to enhance tumor reaction in a general sense without causing serious systemic toxicity.

4. Prolonging life or late-year survival time: the present invention has found experimentally that intermittent treatment with once every two weeks dosing, starting from a certain point in time when WT mice are very old (e.g. 24-27 months old), surprisingly allows for a 72.8% longer median survival after treatment than in the veccle group, with a lower risk of mortality. This finding suggests that SAA-mediated senescent cell clearance can reduce the risk of death in aged mice and effectively prolong their survival.

Based on this, the present invention provides the use of SAA for the preparation of a formulation or pharmaceutical composition for:

a) Inhibiting senescence-associated secretion phenotype (SASP) expression;

b) Anti-aging or anti-aging cells;

c) Reduce the drug resistance of the tumor to the chemotherapeutic drugs;

d) Prolonging the service life or the late-year survival time.

In some embodiments, the reducing the resistance of the tumor to the chemotherapeutic agent is by eliminating senescent cells in the tumor microenvironment and/or inhibiting expression of SASP in the tumor microenvironment. The tumor comprises: prostate cancer, lung cancer, stomach cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof. In a preferred embodiment, the tumor is a tumor which is treated by a genotoxic chemotherapeutic agent, has aging cells increased in the tumor microenvironment and/or highly expresses SASP, and is susceptible to drug resistance to chemotherapy. The "chemotherapeutic agent" is preferably a chemotherapeutic agent capable of inducing an increase in senescent cells and/or a high expression of SASP in the tumor microenvironment.

Pharmaceutical compositions and methods of administration

Based on the synergistic effect of SAA and chemotherapeutic agents discovered in the present invention, the present invention also provides a pharmaceutical composition or kit comprising a therapeutically effective amount of:

c2 A chemotherapeutic agent as a second active ingredient,

wherein the chemotherapeutic agent is capable of inducing the appearance of senescent cells in the tumor microenvironment. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

In the present invention, the chemotherapeutic agent used in combination with SAA is preferably a genotoxic agent capable of inducing the appearance of senescent cells in the tumor microenvironment, more preferably capable of inducing elevated SASP expression in the tumor microenvironment or capable of inducing the senescence marker p16 ^INK4A Up-regulating.

The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredients of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the pharmaceutical composition are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions.

The present invention discovers that the effective amount of SAA can be varied for different uses. For example, in some embodiments, when SAA is used to inhibit SASP expression, its concentration may be 10-200. Mu.M, preferably 20-100. Mu.M, more preferably 40-60. Mu.M (e.g., 40, 45, 50, 55, or 60. Mu.M).

In other embodiments, when SAA is used to induce senescent cells into a death program, its concentration may be > 100. Mu.M, preferably 200-3000. Mu.M, more preferably 200-2000. Mu.M (e.g., 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000. Mu.M).

Also, the effective amount of the active ingredient according to the present invention may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. Generally, satisfactory results are obtained when the active ingredient of the present invention is administered at a dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight) every two weeks. For example, several separate doses may be administered, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Under certain conditions, the frequency of use of senolytic drugs may depend on the rate of accumulation of senescent cells, which may vary depending on the environment in which cell senescence occurs. For example, repeated exposure to cancer therapies that destroy DNA or a sustained high fat diet may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics may reduce the risk of adverse reactions in the patient and allow for use of senolytics during health. Furthermore, intermittent administration can also reduce side effects caused by senolytics and reduce the likelihood of resistance in patients. In contrast to the case of anticancer drugs or antibiotics, since senescent cells do not divide, the body cannot rely on cell proliferation to develop senolytics resistance, and thus cannot obtain favorable mutations, which can create a good basis for the wide use of senolytics in the clinic.

Antitumor substance screening

Based on the combined anti-tumor mechanism of SAA and chemotherapeutic drugs discovered by the invention, the invention provides a method for screening substances which can cooperate with salvianolic acid A to resist tumors. The method may be a screening for known chemotherapeutic agents or may be a screening for agents with an undefined anti-cancer effect. The method screens whether candidate substances are suitable for being cooperated with SAA to resist tumors by detecting the influence of the candidate substances on cell aging in tumor microenvironment.

The screening method of the invention comprises the following steps:

Wherein, the detecting cell aging condition comprises: (i) detecting the number of senescent cells; (ii) detecting SASP factor expression; (iii) Detection of senescence marker p16 ^INK4A . Based on the results of the foregoing studies, it was detected that candidate substance treatment could increase (e.g., increase by 10%, 20%, 30%, 40%, 50% or more) the number of senescent cells, SASP factor expression, and/or the senescence marker p16 in tumor tissue ^INK4A When the salvianolic acid A is combined with the substance, the effect of eliminating aging cells in the tumor microenvironment or reducing SASP factor expression in the tumor microenvironment can be exerted, so that the substance is determined to be a substance which can be synergistic with the salvianolic acid A and can resist tumors.

The screening method of the present invention may be performed by in vitro experiments or in vivo experiments, but is not limited thereto.

The main advantages of the invention include:

1) Provides the application of salvianolic acid A in cell aging, tumor treatment and longevity hit.

2) Provides the combined therapy of the salvianolic acid A and the genotoxic chemotherapeutic drug for resisting tumor, and verifies that the salvianolic acid A and the genotoxic chemotherapeutic drug have obvious synergistic effect.

3) Salvianolic acid A is a medicine which is widely applied clinically and has higher safety.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and fractions are weight percentages and weight fractions unless otherwise indicated.

Materials and methods

1. Cell culture

(1) Maintenance of cell lines

The normal human prostate primary stromal cell line PSC27 (obtained from Research Center, U.S. Fred Hutchinson Cancer) was cultured in an incubator at 37℃under 5% CO2 conditions, propagated and passaged in PSCC complete broth.

(2) Cell cryopreservation and resuscitation

a. Cell cryopreservation

Cells in log phase were collected with 0.25% trypsin, centrifuged at 1000rpm for 2min, the supernatant was discarded and the cells resuspended in freshly prepared frozen stock. The cells were dispensed into labeled sterile cryopreservation tubes. Then cooling by gradient, and finally transferring into liquid nitrogen for long-term storage.

b. Cell resuscitation

The frozen cells in the liquid nitrogen are taken out and immediately put into a water bath with the temperature of 37 ℃ to be quickly melted. Directly adding 2 ml cell culture solution to make the cells uniformly suspended. After the cells adhere to the wall, a new culture solution is replaced.

(3) In vitro experimental treatment

To cause cell damage, 50. Mu.g/ml Bleomycin (BLER) was added to the culture broth when PSC27 cells grew to 80% (PSC 27-CTRL for short). After 12 hours of drug treatment, the cells were briefly washed 3 times with PBS, left in culture for 7-10 days, and then subjected to subsequent experiments.

2. Screening of natural product libraries

Pharmacodynamic analysis was performed against a pool of organic chemical drugs (TOPSCIENCE) which contains 1470 small molecule compounds in total, is mostly molecules of medicinal plant origin and has anti-aging potential. The various products were diluted to 96-well plates at a density of 5000 cells per well, respectively, according to a concentration gradient. The working concentration of the natural product (or compound) is typically controlled at 1. Mu.M-l mM using DMEM for the medium. 3-7 days after drug treatment, cell proliferation was determined using CCK-8Cell Counting Kit kit (based on WST-8 principle, vazyme) and apoptotic activity was determined as Caspase 3/7activity kit (Promega).

The initially identified drug candidates were screened for a further 30 days. Drugs entering the second round candidate range were diluted into 6-well plates with 20,000 cells per well. The medium and drug candidate were changed every other day. To determine the effect of each drug on cell phenotype and viability, etc., the project was validated against different concentrations of drug.

3. Immunoblotting and immunofluorescence detection

Cell lysis derived proteins were separated by NuPAGE 4-12% bis-Tris gel and transferred onto nitrocellulose membrane (Life Technologies). Blotting was blocked with 5% skim milk at room temperature for 1h, incubated with the required primary antibody at 4℃overnight at the manufacturer's protocol, then with horseradish peroxidase-conjugated secondary antibody (Santa Cruz) at room temperature for 1h, membrane blotting signaling was performed with Enhanced Chemiluminescence (ECL) detection reagents (Millipore) according to the manufacturer's protocol, and using ImageQuant LAS 400 Phospho-Imager (GE Healthcare). As a standard protein marker, pageRuler Plus Prestained Protein Ladder (No. 26619) provided by Thermo Fisher Scientific company was used by the present inventors.

For immunofluorescent staining, target cells were pre-seeded on the coverslide for at least 24h after being cultured in a petri dish. After brief washing, fixation with 4% paraformaldehyde in PBS was performed for 8min, and blocking with 5% normal goat serum (NGS, thermo Fisher) was performed for 30min. Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser 139) (clone JBW301, millipore) and mouse monoclonal antibody anti-BrdU (Cat# 347580,BD Biosciences), and secondary antibody Alexa 488 (or 594) -conjugated F (ab') 2 was sequentially added to the fixed cell-covered slide. The nuclei were counterstained with 2. Mu.g/ml of 4', 6-diamidino-2-phenylindole (DAPI). And selecting a most representative image from the 3 observation fields for data analysis and result display. FV1000 laser scanning confocal microscope (Olympus) was used to acquire cell confocal fluorescence images.

4. Whole transcriptome sequencing analysis (RNA-sequencing)

Full transcriptome sequencing was performed on the human prostate primary stromal cell line PSC27 under different processing conditions. Total RNA samples were obtained from stromal cells. The integrity was verified by Bioanalyzer 2100 (Agilent), RNA was sequenced with Illumina HiSeq X10 and gene expression levels were quantified by software package rsem (https:// deweylab. Github. Io/rsem /). Briefly, rRNA in RNA samples was eliminated with RiboMinus Eukaryote kit (Qiagen, valencia, CA, USA); and a strand-specific RNA-seq library was constructed with TruSeq Stranded Total RNA preparation kits (Illumina, san Diego, calif., USA) prior to deep sequencing according to the manufacturer's instructions.

Paired-end transcriptomic reads was mapped to the reference genome (GRCh 38/hg 38) and annotated with reference from Gencode v27 using the Bowtie tool. Repeated reads are identified using picard tools (1.98) script tag repeat items (https:// github. Com/broadenstite/picard), leaving only non-repeated reads. Reference splice junctions is provided by the reference transcriptome (Ensembl build 73). And (3) calculating an FPKM value by using Cufflinks, and calling differential gene expression by using the Cufflinks and a maximum likelihood estimation function. Genes expressing significant changes were defined by False Discovery Rate (FDR) -corrected P value <0.05, and were analyzed downstream using only state "Known" and biological "coding" ensembl genes 73.

The Reads were then trimmed using Trim Galore (v0.3.0) (http:// www.bioinformatics.babraham.ac.uk/projects/trim_galore /), while the quality assessment used FastQC (v0.10.0) (http:// www.bioinformatics.bbsrc.ac.uk/projects/FastQC /). Subsequently, DAVID bioinformatics platform (https:// david. Ncifcrf. Gov /), ingenuity Pathways Analysis (IPA) program (http:// www.ingenuity.com/index. Html) was used. Preliminary analysis was performed on the raw data on a Majorbio I-Sanger Cloud Platform (www.i-sanger.com) free online platform, and the raw data was stored in a NCBI Gene Expression Omnibus (GEO) database under the registration code GSE156448.

5. Protein-protein interaction network analysis

Protein-protein interaction (PPI) analysis was performed with sting 3.0. Specific proteins meeting the standards are imported into on-line analysis software (http:// www.networkanalyst.ca), and a minimum interaction network is selected for further hub and module analysis.

6. Gene Set Enrichment Analysis (GSEA)

Based on the data obtained from the RNA-seq preliminary analysis, for each differential expression significant gene analysis comparison, genes were ranked using "wald statistics" obtained from DESeq2, and GSEA was performed on these ranked lists of all the planned gene sets available in MSigDB (http:// software.broadinstall.org/GSEA/MSigDB). DESeq2 independent filtering was based on the average of normalized read counts to screen for genes with very low expression levels. SASP and GSEA signature are described in the published literature by the inventors (Zhang et al, 2018 a).

7. Quantitative PCR (RT-PCR) determination of Gene expression

(1) Extraction of total RNA from cells

Extracting total RNA of cells in a growing period or a stopping period by using a Trizol reagent, adding 1ml of Trizol into each T25 culture flask cell, scraping a cell layer by using a cell scraper, transferring the cell layer into a centrifuge tube, and fully and uniformly mixing the cell layer until the cell layer is not sticky. Adding 0.2ml chloroform into each 1ml Trizol, shaking vigorously for 15sec, and incubating at room temperature for 5-10min; centrifuging at 4 ℃ for 15min at 11,000 g; transferring the colorless supernatant into a new centrifuge tube, adding 0.5. 0.5 ml isopropanol to each 1ml of Trizol, incubating for 10 minutes at room temperature, centrifuging for 10 minutes at 11,000g and 4 ℃; the supernatant was decanted, washed with 75% ethanol (at least 1ml of 75% ethanol per 1ml of Trizol), and centrifuged at 7,500g for 5min at 4 ℃; drying RNA precipitate at room temperature for 5-10min (RNA cannot be dried), and drying with DEPC-H ₂ O dissolves the precipitate.

After quantifying RNA by a spectrophotometer, a small amount of total RNA was subjected to 1% agarose electrophoresis, and the RNA state and quality were checked.

(2) Reverse transcription reaction

OligodT ₂₃ V _N (50uM) 1ul

Total RNA 1-2ug

RNase Free ddH ₂ O to 8ul

Heated at 65℃for 5min, rapidly quenched on ice and allowed to stand for 2min.

Preparing first strand cDNA Synthesis solution

2x RT Mix 10ul

HiScript II Enzyme Mix 2ul

First strand cDNA synthesis was performed according to the following conditions:

25℃ 5min

50℃ 45min

85℃ 5min

(3) Real-time quantitative PCR reaction

The cDNA of the reverse transcription reaction product was diluted 50-fold as a template.

Sample addition according to the above standard, the reaction conditions are: pre-denaturation at 95℃for 15sec, followed by 5sec at 95℃for 31sec at 60℃for 40 cycles; the melting curve conditions were 95℃for 15sec,60℃for 30sec, and 95℃for 15 sec. Samples were reacted on an ABI ViiA7 (ABI) instrument. The expression of beta-actin is used as an internal reference. After the reaction is completed, the amplification condition of each gene is checked through software analysis, the corresponding threshold cycle number is derived, and the relative expression quantity of each gene is calculated by adopting a 2-delta Ct method. Peaks and waveforms of melting curves (mering curves) were analyzed to determine if the resulting amplified product was a specific single fragment of interest.

SA-beta-Gal staining

Aging-related beta-galactosidase (SA-beta-Gal) staining was performed by conventional procedures (Debacq-Chainiaux et al, 2009). Briefly, the cell culture dishes were washed with PBS and fixed at room temperature. The cells were fixed by 3min in 2% formaldehyde and 0.2% glutaraldehyde. SA- β -Gal was then stained with freshly prepared staining solution at 37℃overnight. The next day an image was taken and the percentage of positive cells per unit area was calculated.

9. Cloning amplification experiments

Single cell cloning experiments were performed as described in the past literature (Duan et al, 2015; wu et al, 2018). Briefly, cells were plated in gelatin coated 12-well plates at a density of 2000 cells/well. Cell clone numbers were counted after crystal violet staining.

10. Drug-induced aging cell apoptosis

PSC27 cells were plated in 96-well dishes and cell senescence was induced under 50 μg/ml BLER treatment. SAA and ABT263 were added at 200. Mu.M and 1.25. Mu.M concentrations, respectively. Cell culture medium was supplemented with Incucyte Nuclight rapid red reagent (Essen Bioscience) and IncucyteC-3/7 apoptosis reagent (Essen Bioscience). Representative fields of view were selected for photographing.

11. Inoculation and preclinical treatment test of mouse transplanted tumor

All experimental mouse experiments were performed strictly following the regulations of the national academy of sciences Shanghai life sciences laboratory animal Care and use Committee (IACUC). Immunodeficient mice (NOD-SCID mice, ICR) (weighing about 25 g) aged 6-8 weeks were used in the patent related animal experiments. Stromal cells PSC27 and epithelial cells PC3 were mixed in a predetermined ratio of 1:4, and each transplant contained 1.25X10 ⁶ Cells for tissue reconstruction. The transplanted tumor was implanted into mice by subcutaneous implantation, and animals were euthanized 8 weeks after the implantation procedure was completed. Tumor volume was calculated according to the following formula: v= (pi/6) x ((l+w)/2) ³ (V, volume; l, length; w, width).

In pre-clinical treatment trials, subcutaneously transplanted mice were fed standard experimental diet and 2 weeks later were given intraperitoneal administration of the chemotherapeutic drugs mitoxantrone (MIT, 0.2mg/kg dose) and/or SAA (500 μl,10mg/kg dose). The time points are as follows: the former on the first day of weeks 3,5,7 and the latter on the first day of weeks 5, 7. The whole course of treatment was followed by 3 MIT cycles of 2 weeks each. After the end of the course, the mouse tumors were collected for volume measurement and histological analysis. Each mouse cumulatively received MIT as a drug at 0.6 mg/kg body weight, while SAA was 30mg/kg body weight. To cause systemic SASP factor expression under chemotherapy induction, MIT was administered to mice by intravenous infusion in the above steps and sequences, but the dose was reduced to 0.1mg/kg body weight per time (cumulative MIT dose received for the entire course of treatment was 0.3mg/kg body weight) to reduce drug-related toxicity. Chemotherapy experiments were performed to the end of week 8, and mice were dissected immediately after sacrifice, and their transplanted tumors were collected and used for pathological system analysis.

12. Mouse longevity study

In cell transplantation studies, the inventors obtained 16 month old male C57BL/6 mice on SPF animal platforms by continuous feeding, with 4 to 5 animals per cage. The inventors first classified mice from low to high body weight and then selected mice with similar body weight. Next, either the Senescent (SEN) or Control (CTRL) transplant treatment regimen was assigned to mice at each interval using a random number generator, while the middle mice were assigned to another treatment regimen to match the body weights of the senescent and control transplanted mice. After 1 month of cell transplantation, body function tests were performed when the mice were 18 months of age. After that, no further tests were performed on these mice, except for checking their cages. The earliest death occurs approximately 2 months after the last physical function test. C57BL/6 mice 19 to 21 months old, 3-5 mice were housed in each cage. As with the transplanted mice, the mice were classified according to body weight and randomly assigned to each group, and treated with a control group (vehicle) or a drug group (SAA) group by persons who did not know the design of the pre-clinical trial. Starting from 24-27 months of age, mice were treated with vehicle or SAA every 2 weeks, each for 3 consecutive days, orally, by gavage. During the course of the study, some mice were removed from the original cages to try to avoid the stress of living animals in a single cage caused by long-term rearing. The RotaRod and ranging tests are performed monthly because these tests are sensitive and non-invasive. At the end of the trial, the inventors euthanized mice; the inventors consider that they have died if they exhibit one of several symptoms: (one) cannot drink water or eat; (II) is not willing to move even if stimulated; (III) fast weight loss; (IV) serious balance disorder; or (V) bleeding or ulceration of the body. During the test, no mice were excluded from the test due to cradling, accidental death or dermatitis. When performing biological statistics, the inventors used Cox proportional hazard model to conduct survival analysis.

13. Preclinical post-mortem pathological examination of animals

The investigator examined the cages daily and removed the dead mice from the cages. Within 24 hours of animal death, cadavers were opened (abdominal, thoracic, and skull) and kept alone in 10% formalin for at least 7 days. The decomposed or destroyed body is excluded. The preserved cadavers were transported to an Autopsy dedicated site for pathological examination. Tumor burden (sum of different types of tumors per mouse), disease burden (sum of different histopathological changes of major organs per mouse), severity of each lesion and inflammation (lymphocyte infiltration) were evaluated.

14. Bioluminescence imaging

Mice were intraperitoneally injected with 3mg fluorescein (BioVision, milpitas, calif.) delivered in 200 μl volume of PBS. Mice were anesthetized with isoflurane and bioluminescence images were acquired using a Xenogen IVIS 200System (Caliper Life Sciences, hopkinton, MA).

15. Physical fitness test

All assays started on day 5 after the last placebo or drug treatment. Maximum walking speed was assessed using an accelerated rotation System (TSE System, chesterfiled, MO). Mice were trained on RotaRod for 3 days at speeds of 4,6 and 8 r.p.m., days 1, 2 and 3 for 200 seconds, respectively. On the test day, mice were placed on RotaRod, starting at a speed of 4 r.p.m. At 5 minute intervals, the rotational speed was accelerated from 4 to 40r.p.m. When the mouse falls from RotaRod, the velocity is recorded. The final results were averaged from 3 or 4 trials and normalized to baseline speed. Mice trained during the first two months were no longer trained.

Forelimb grip strength (N) was determined using Grip Strength Meter (Columbus Instruments, columbus, OH) and results from an average of more than 10 trials. For the suspension endurance test, the mice were placed on a 2 mm thick wire, the latter being located 35 cm above the mat. The mice were allowed to grasp the wires with the forelimbs only, and the suspension time was normalized to the body weight, expressed as suspension duration (sec) x body weight (g). The results were averaged from 2 to 3 experiments per mouse. Daily activity and food intake were monitored by Comprehensive Laboratory Animal Monitoring System (CLAMS) for 24 hours (12 hours light and 12 hours dark). The CLAMS system is equipped with Oxymax Open Circuit Calorimeter System (Columbus Instruments). For treadmill performance, mice were adapted to run on an electric treadmill (Columbus Instruments) at a 5 ° incline, trained for 3 days, for 5 minutes per day, starting at a speed of 5 meters/minute for 2 minutesFollowed by acceleration to 7 m/min for 2 min and then 9 m/min for 1 min. On the day of the test, the mice were run on the treadmill for 2 minutes at an initial speed of 5 meters/min, then every 2 minutes at an increased speed of 2 meters/min until the mice were exhausted. Fatigue is defined as the inability of a mouse to return to the treadmill even with mild shock and mechanical stimulation. The distance was recorded after the end of the test and the total work (KJ) was calculated using the following formula: mass (kg). Times.g (9.8 m/s) ² ) X distance (m) ×sin (5 °).

16. Biometric method

All in vitro experiments involving cell proliferation rate, viability and SA- β -Gal staining, etc. and in vivo experiments with mouse transplantations and pre-clinical drug treatment in this patent application were repeated more than 3 times, and the data were presented as mean.+ -. Standard error. Statistical analysis was based on raw data calculated by one-way analysis of variance (ANOVA) or a two-measured Student's t-test, while the results of P <0.05 were considered to have significant differences.

Correlation between factors was examined with Pearson's correlation coefficients. When mice were obtained in several queues and grouped in cages, a survival analysis was performed using Cox proportional hazard model. The model treats gender and age of treatment as a fixed effect, and queue and initial cage assignment as a random effect. Since some mice were removed from the original cage during the study to minimize stress from the single cage housing, the inventors also performed an analysis without the cage effect. The results of these two analyses are not greatly different in directionality or statistical sense, enhancing confidence in the results of the present inventors. Survival analysis used statistical software R (version 3.4.1; library 'coxme'). In most experiments and outcome evaluations, the investigator resorted to blind selection of the assignments. The inventors assigned mice to experimental groups using baseline body weights (to achieve similar body weights between groups), so randomization was performed only within groups matched to body weights. The inventors determined the sample size based on previous experiments, and therefore did not use statistical power analysis. All replicates in this study were from different samples, each from a different experimental animal.

EXAMPLE 1SAA is effective in inhibiting SASP expression when used at low concentrations

To identify novel compounds that are effective in modulating the phenotype of senescent cells, the present inventors have developed unbiased screens using an organic chemical library consisting of 1470 small molecules. To examine the efficacy and potential biological value of these drugs, the inventors selected the use of the primary normal human prostate stromal cell line, PSC27, as an in vitro cell model. PSC27 is composed primarily of fibroblasts, but not fibroblast lines (including endothelial cells and smooth muscle cells) are also present, but in smaller proportions, PSC27 is a primary stromal cell line of human origin in nature, forming typical SASPs upon exposure to stress factors such as genotoxic chemotherapy (genotoxic chemotherapy) or ionizing radiation (ionizing radiation). The inventors treated these cells in a manner that had been optimized in the preliminary experiments, i.e. with a specific dose of Bleomycin (BLEO), and observed a significant increase in the staining positive rate of the aging-related β -galactosidase (SA- β -Gal), a significant decrease in BrdU incorporation efficiency, and a significant increase in DNA damage repair foci (DDR foci) within days after drug damage (fig. 1-3). The inventors compared the effect of these natural drug products on senescent cell expression profiles in parallel by means of high-throughput high-inclusion system screening (fig. 4).

The inventors performed RNA-seq sequencing on these cells. The high throughput data obtained subsequently shows that a small molecule compound, salvianolic Acid A (SAA) (fig. 5), significantly alters the expression profile of senescent cells. Wherein significant downregulation of thousands of genes occurs while upregulation of multiple genes occurs. Importantly, the expression of SASP factors in senescent cells after SAA treatment was generally reduced, whereas these SASP factors were generally significantly up-regulated in senescent cells (FIG. 6). While the expression of some SASP-unrelated genes showed similar trends to those typical SASP factors, the data from GSEA analysis further revealed significant inhibition of molecular tags that characterize SASP expression or NF-KB activation, the latter being the major transcriptional event mediating the development of pro-inflammatory SASP (FIG. 7, FIG. 8). Further GO bioinformatics data indicate that these molecules are functionally involved in a set of important biological processes including signal transduction, intercellular communication, cellular metabolism, energy modulation, nucleotide metabolism, and protein metabolism (fig. 9). Most of these down-regulated genes, apart from playing a role in the nucleus, are essentially proteins that are released into the extracellular space upon expression, or are located on the plasma membrane or in the cytoplasm, and generally characteristically interact with the secretory properties of these molecules (fig. 10).

To further demonstrate the effect of SAA on SASP expression under in vitro conditions, the inventors treated PSC27 cells under a range of in vitro concentration gradients. The data indicate that SAA at a working concentration of 50. Mu.M inhibited SASP development with maximum efficiency (FIG. 11). However, the efficacy of such drugs at lower or higher concentrations is not ideal, although the latter may be associated with cellular stress response caused by increased cytotoxicity of such drugs (fig. 11). Thus, SAA, a natural small molecule compound of plant origin, can be used to control the pro-inflammatory phenotype of senescent cells, i.e., SASP, particularly at relatively low concentrations.

Example 2 SAA is a novel senolytics when used at high concentrations

In view of the remarkable therapeutic efficacy of SAA in controlling SASP expression, the inventors next explored the potential of this natural product to kill senescent cells at higher concentrations. To this end, the inventors measured the percentage of senescent cells treated under in vitro conditions as the SAA concentration increased. SA- β -Gal staining data showed that senescent cells were not substantially eliminated until SAA concentration reached 200. Mu.M (FIG. 12). With increasing concentration, the killing effect of SAA on senescent cells (80% positive staining) was further enhanced, whereas the threshold was reached when SAA was at 2000. Mu.M (senescent cells remained at about 20%); when its concentration was raised to 3000. Mu.M, the killing effect of SAA was not further enhanced (FIG. 12; FIG. 13).

To further address these issues, the inventors have made a validation experiment. Cell viability assays showed that SAA induced significant death of senescent cells starting at a concentration of 200 μm compared to their proliferating control cells (fig. 14). When the SAA concentration was increased to 2000. Mu.M, the percentage of surviving senescent cells was reduced to about 10%. However, proliferation cells were not significantly reduced even at 3000 μm of SAA. These results demonstrate the high selectivity and outstanding specificity of SAA for senescent cells, a feature which is actually the basic technical requirement for senolytics as a unique class of anti-aging agents.

The inventors next investigated the potential of population doubling (population doubling, PD) of stromal cells after genotoxic treatment. The combination treatment group of BLEO and SAA showed significantly increased PD capacity compared to the group of cells that entered the growth arrest state immediately after the damaging treatment (fig. 15). Interestingly, however, SAA by itself did not appear to affect PD in proliferating cells, and this data further suggests that SAA is selective between senescent and normal cells.

To investigate whether SAA causes senescent cells to lose viability by inducing apoptosis, the present inventors treated proliferation and senescent cells separately under culture conditions with SAA. The subsequent observed changes in caspase-3/7 activity indicate that SAA causes apoptosis in senescent cells; from the 16 th hour after SAA addition, statistical differences between the aged and control groups began to appear (FIG. 16). In addition, the pan caspase inhibitor QVD prevents the killing of senescent cells by SAA, a process that has a practical effect very similar to that of ABT263, a currently known, very effective inducer of apoptosis in senescent cells (FIG. 17). The above series of results demonstrate that SAA promotes entry of senescent cells into the death program by inducing apoptosis, but that proliferating cells are not substantially targeted or affected by this natural drug.

Given the apparent impact of SAA on senescent cells, the inventors subsequently analyzed the potential of SAA to induce apoptosis. Flow cell data showed that senescent PSC27 cells were significantly reduced in viability and their proportion of apoptosis was significantly increased, but the changes in proliferating cells were not apparent (fig. 18, fig. 19). Thus, the data consistency of the present invention supports that SAA causes the elimination of senescent cells by inducing apoptosis under in vitro conditions, a natural product that has outstanding potential in targeting senescent cells.

Example 3 therapeutic targeting of senescent cells with SAA promotes tumor regression and effectively reduces chemotherapy resistance

In view of the outstanding selectivity of SAA in eliminating senescent cells at higher concentrations in vitro, the inventors next considered whether this drug could be used to interfere with a variety of diseases associated with aging in vivo. In many human pathologies, cancer is one of the major chronic diseases that severely threatens human life and endangers health. Furthermore, cancer cell resistance in the clinic limits the effectiveness of most anti-cancer therapies, whereas senescent cells often promote the development of therapeutic resistance to their peripheral cancer cells by developing SASP in damaged foci. Even so, the feasibility and safety of eliminating senescent cells from primary tumors to promote cancer therapeutic index has been hardly explored by scientists to date.

First, the present inventors constructed tissue recombinants, which are a typical highly malignant prostate cancer cell line, by mixing PSC27 stromal cells with PC3 epithelial cells. The number ratio of stromal cells to epithelial cells prior to subcutaneous implantation of recombinants in the rear thigh of non-obese diabetic and severe combined immunodeficiency (NOD/SCID) experimental mice was 1:4. animals were measured for tumor size (volume) at the end of 8 weeks after recombinant implantation (fig. 20). The xenograft (xenograft) consisting of PC3 cells and senescent PSC27 cells had significantly increased volume (P < 0.001) compared to tumors consisting of PC3 cancer cells and primary PSC27 stromal cells, this difference again confirming the key promoting role of senescent cells in tumor progression (fig. 21).

To technically more closely approximate clinical conditions and related background, the present inventors devised a preclinical protocol specifically involving genotoxic chemotherapeutic drug treatment and/or senescent drug intervention (fig. 22). After two weeks of subcutaneous implantation, when it was observed that the tumor had been stably ingested in vivo, the inventors provided a single dose of MIT (MIT; a chemotherapeutic agent) or placebo, respectively, to the experimental animals on the first day of weeks 3, 5 and 7 until the 8 week regimen was all ended. The administration of MIT significantly delayed tumor growth compared to placebo-treated group, which demonstrated the efficacy of MIT as a chemotherapeutic drug (45.9% reduction in tumor size, P < 0.0001) (fig. 23). Notably, although SAA does not cause tumor shrinkage by itself, administration of SAA significantly reduced tumors in mice following MIT treatment (39.9% reduction in tumor volume compared to MIT, P <0.01; 67.5% reduction in tumor volume compared to placebo treatment, P < 0.0001) (fig. 23).

Next, the inventors inferred whether cellular senescence occurred in the foci of these animals. The results of the test demonstrate that the MIT dosing process induces the appearance of a large number of senescent cells in tumor tissue, although this seems not to be surprising. However, SAA administration substantially depletes most of the senescent cells within the lesions of these chemotherapeutics (FIG. 24; FIG. 25). Laser Capture Microdissection (LCM) and subsequent quantitative PCR results showed a significant increase in SASP factor expression, including IL6, CXCL8, SPINK1, WNT16B, GM-CSF, MMP3, IL1 a, a trend that was accompanied by a chemotherapeutical animal senescence marker p16 ^INK4A Up-regulation of (fig. 26). Interestingly, these changes occur primarily in stromal cells, rather than in their neighboring cancer cells, which means the possibility of repopulating residual cancer cells, which create acquired resistance in the TME for the treatment of lesions. However, this change was largely reversed when SAA was administered, as demonstrated by the results of the transcript level data analysis (FIG. 27).

To investigate the mechanism directly supporting the expression of SASP and reversing this senescence-associated pattern in MIT-dosed mice, the inventors dissected tumors in animals treated with both drugs 7 days after the first SAA administration, which time point after 7 days of administration was chosen mainly because cancer cell resistant clones in the lesions had not yet formed. MIT administration resulted in a significant increase in both DNA damage and apoptosis compared to placebo. Although SAA alone was unable to induce DNA damage or cause apoptosis, chemotherapeutic drug MIT could highly up-regulate both of these indicators (fig. 28). However, when MIT treated animals were used with SAA, the index of DNA damage or apoptosis was significantly increased, which means that cytotoxicity of tumor sites in animals under these aging drug treatment conditions was increased. As supportive evidence, caspase 3 clear activity was elevated when SAA was applied during treatment, a typical marker of apoptosis (fig. 29).

Next the inventors compared the survival of animals from different drug treatment groups, mainly to evaluate the outcome of tumor progression in a time-prolonged manner. In this preclinical cohort, the inventors performed tumor growth monitoring on animals, once the mice had a prominent endosomal tumor burden (size ≡2000mm ³ ) It is judged that serious diseases have occurred, which is a method for the progress of diseases such as tumors in some cases. Mice treated with the MIT/SAA combination exhibited the longest median survival, which was prolonged by at least 48.1% compared to the group treated with MIT alone (fig. 30, cyan versus blue). However, treatment of tumor-bearing mice with SAA alone did not result in significant benefit, with only marginal survival extension.

Notably, the treatments performed in these studies appeared to be well tolerated by the experimental mice. The inventors did not observe significant fluctuations in urea, creatinine, liver enzymes or body weight (fig. 31; fig. 32). More importantly, the chemotherapy and anti-aging drugs used at each drug dose designed in this study did not significantly interfere with the integrity of the immune system and tissue homeostasis of critical organs, even in immunized intact wild-type mice (fig. 33; fig. 34). These results consistently demonstrate that anti-aging agents in combination with conventional chemotherapeutic agents are likely to enhance tumor response in a general sense without causing severe systemic toxicity.

To determine whether SAA has drug dependence or specificity in enhancing the efficacy of chemotherapy, the inventors then chose to use Doxorubicin (DOX), docetaxel (DOC), and Vincristine (VIN), in combination with SAA, respectively, for pre-clinical trials. The results indicate that only DOX in combination with SAA can substantially repeat the significant effects of MIT in combination with SAA in these chemotherapeutics (fig. 35). While DOC and VIN, although able to reduce tumor volume when used alone, did not cause tumor further shrinkage when SAA was co-administered with it, i.e., failed to deliver further benefit (fig. 36, fig. 37). Therefore, SAA has the characteristic of improving the therapeutic effect of chemotherapy under in vivo conditions, is limited to the combination with specific genotoxic drugs, and has drug type dependence.

EXAMPLE 4 senescent cell clearance by SAA treatment can extend the later-year survival of experimental mice

Based on the surprising efficacy of SAA in eliminating senescent cells, reducing tumor resistance and improving overall therapeutic efficacy in the microenvironment of tumor mice, the present invention further investigated whether it also has some significant health promoting or disease delaying benefit in naturally senescent animals. The inventors first considered whether a potentially transforming method could be used to eliminate senescent cells, namely: intermittent treatment was performed starting from a very old time point and it was investigated whether the residual life of WT mice could be prolonged. Based on this, a series of in vivo tests were performed. Surprisingly, the median survival after treatment of the SAA group, given starting from 24-27 months of age (corresponding to the age of 75-90 years of human) under a treatment regimen with once every two weeks of administration, was prolonged by 72.8% compared to the Vehicle group, with a lower risk of mortality (HR=0.33, SAA/Vehicle group; P < 0.0001) (FIG. 38, FIG. 39). This finding suggests that SAA-mediated senescent cell clearance can reduce the risk of death in aged mice and effectively prolong their survival.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. Use of salvianolic acid a for the preparation of a formulation or pharmaceutical composition for:

a) Inhibiting senescence-associated secretion phenotype (SASP) expression;

b) Anti-aging or anti-aging cells;

D) Prolonging the service life or the late-year survival time.

2. Use according to claim 1, wherein the concentration of salvianolic acid a in the preparation or pharmaceutical composition for inhibiting the expression of SASP is 10-200 μm, preferably 20-100 μm, more preferably 40-60 μm (e.g. 40, 45, 50, 55 or 60 μm).

3. Use according to claim 1, wherein the concentration of salvianolic acid a in the preparation or pharmaceutical composition for eliminating senescent cells is > 100 μm, preferably 200-3000 μm, more preferably 200-2000 μm (e.g. 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μm).

4. The use of claim 1, wherein the chemotherapeutic agent is selected from the group consisting of: mitoxantrone, bleomycin, doxorubicin, or a combination thereof.

5. The use of claim 1, wherein the neoplasm is selected from the group consisting of: prostate cancer, lung cancer, stomach cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof.

6. A pharmaceutical composition or combination comprising a therapeutically effective amount of:

c2 A chemotherapeutic agent as a second active ingredient,

7. As claimed inThe pharmaceutical composition or combination of claim 6, wherein the chemotherapeutic agent is capable of inducing SASP expression in a tumor microenvironment or is capable of inducing the senescence marker p16 ^INK4A Up-regulating.

8. A method for screening a substance capable of synergistically resisting tumor with salvianolic acid a, comprising the steps of:

9. The method of claim 8, wherein said detecting a cellular aging condition comprises:

(i) Detecting the number of senescent cells;

(ii) Detecting SASP factor expression;

(iii) Detection of senescence marker p16 ^INK4A 。

10. A method of non-therapeutically inhibiting cellular SASP expression, clearing senescent cells, and/or reducing resistance of tumor cells to a chemotherapeutic agent in vitro comprising the steps of:

contacting salvianolic acid a with said cells, thereby inhibiting its SASP expression, clearing said senescent cells, and/or reducing the resistance of said tumor cells to chemotherapeutic agents.