CN111228266A

CN111228266A - Application of GW8510 in preparation of medicines for prolonging life and improving cognitive ability of mammals during natural aging

Info

Publication number: CN111228266A
Application number: CN202010156556.5A
Authority: CN
Inventors: 谢正伟; 安永盼; 杨宝学; 朱杰
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-06-05
Anticipated expiration: 2040-03-09
Also published as: WO2021179952A1; CN111228266B

Abstract

The invention provides an application of GW8510 in preparing a medicine for prolonging the life of a mammal in natural aging, improving cognitive ability and/or improving muscular ability and/or improving motor ability, and in vitro mechanism research shows that the GW8510 can regulate and control the expression of cell cycle related protein p21 and CDK family genes, so that the replicative aging state of a natural aging cell line 2BS and WI38 is improved, and the mitochondrial function of an aging cell can be improved; mouse experiments show that GW8510 can prolong the lifespan of normal aging mice.

Description

Application of GW8510 in preparation of medicines for prolonging life and improving cognitive ability of mammals during natural aging

Technical Field

The invention belongs to the field of biology, and particularly relates to application of GW8510 in preparation of medicaments for prolonging the natural aging life of mammals and improving cognitive ability.

Background

Aging is a process of the degeneration of the functions of tissues and organs of an organism with the aging, and is closely related to the normal embryonic development, the aging of the organism, aging-related diseases and the like. In recent years, the research on cell aging is more and more intensive, and the biological effect of the cell aging on aging resistance is more and more important. Research shows that cell aging (cellular senescence) refers to that the normal physiological function and proliferation capacity of cells gradually decline along with the lapse of time or when the cells face external stress pressure, so that the cells are separated from the cell cycle, and the process has important relation with tumor, tissue regeneration, organism aging and the like. Since the theory of mitochondrial senescence has been proposed, mitochondria have been the focus of senescence research. Mitochondrial function gradually diminishes with age, while cellular degeneration is caused by Reactive Oxygen Species (ROS) with age, where mitochondrial senescence theory regards mitochondria as the major generator of ROS. At present, GW8510 research is still few, and existing research shows that GW8510 has the functions of reducing chemotherapy side effects and preventing alopecia and mucositis.

Disclosure of Invention

The present invention provides for the use of GW8510 in order to prolong the life of mammals as they age naturally.

In a first aspect of the invention, there is provided the use of GW8510 in the manufacture of a medicament for prolonging the life, improving cognitive ability and/or improving muscular ability and/or improving motor ability of a mammal in its natural aging.

Preferably, GW8510 is administered at a dose of 0.22 mg/kg/day to 1.1 mg/kg/day.

In a second aspect of the invention, there is provided the use of GW8510 in the manufacture of a medicament for inhibiting the expression of cyclin-related p21 and/or CDK family genes.

In a third aspect of the invention, there is provided the use of GW8510 in the manufacture of a medicament for the treatment and/or amelioration of mitochondrial function of senescent cells.

In a fourth aspect of the invention, there is provided the use of GW8510 in the manufacture of a medicament for prolonging the lifespan of a replicating senescent cell line.

Preferably, the senescent cell line comprises 2BS, WI 38.

Has the advantages that: the GW8510 provided by the invention can obviously improve the healthy aging of naturally aging mice and prolong the service life of the naturally aging mice, and in vitro mechanism research shows that the GW8510 can regulate and control the expression of cell cycle-related protein p21 and CDK family genes, thereby improving the replicative aging state of naturally aging cell lines 2BS and WI 38. It also can improve mitochondrial function of senescent cells, thereby slowing aging.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 shows that GW8510 can extend budding yeast longevity.

FIG. 2 shows that GW8510 can alleviate replicative senescence of 2BS (PD45) and WI38(PD45) cells; among them, GW8510 analyzes the viability of aged 2bs (a) and WI38(B) cells. Effect of 2bs (c) cell proliferation at PD 45. Mean ± sem.n-5-8. P < 0.05; p < 0.01; p < 0.001.

FIG. 3 shows that GW8510 can improve the cell senescence morphology of aged WI-38, wherein (A) cell senescence SA- β -gal staining (magnification x 400), (B) the amount of change in cell senescence marker P21 gene expression after administration, and (C) the amount of change in cell senescence-associated secretory phenotype (SASP) mRNA expression after administration, Mean + -SEM.n.5-8. P < 0.05;. P < 0.01. P < 0.001.

FIG. 4 shows that GW8510 can affect cellular mitochondrial function and cycle-related gene expression; wherein, (a) cell mitochondrial membrane potential staining (TMRM); (B) mitochondrial membrane potential staining histogram; (C) cellular ATP production; (D) change in cell cycle-associated gene mRNA expression. Mean ± sem.n-5-8. P < 0.05; p < 0.01; p < 0.001.

FIG. 5 shows that GW8510 can prolong the lifespan of naturally aging mice; dosing intervention was initiated at 18 months of age using ICR mice.

FIG. 6 shows that GW8510 can prolong the healthy lifespan of naturally aging mice; wherein C57 mice were used to begin dosing for 16 months (a) novel object recognition experiments (characterization of cognitive ability); (B) grip test (characterization of muscle capacity); (C) fatigue rotameter experiments (characterizing motor ability); (D) y-maze experiment (characterization of cognitive ability).

FIG. 7 shows that GW8510 acts on PAK 1; wherein (A-D) a life curve under intervention of a yeast gene knockout strain GW 8510; (E) ste20(PAK1 yeast homolog) knockout strain life curve; (F) GW8510 and PAK1 protein SPR (surface plasmon resonance) experiments.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

ICR mice, male, 8 weeks, 20-22 g, purchased from the laboratory animal center of the department of medicine, Beijing university.

Maintaining the feed: purchased from the laboratory animal center of the department of medicine of Beijing university.

C57BL/6J mice, male, 13 months, 25-28 g, purchased in Peking sbeful laboratory animal center.

All animal experiments were strictly in accordance with the guidelines for the management and use of laboratory animals and were approved by the North Committee for the management of laboratory animals. During the sacrifice of the animals, the animals were anesthetized and the pain was minimized.

2BS cells (human diploid fibroblasts), WI-38 cells (human embryonic lung fibroblasts) were purchased from the Chinese food and drug testing institute.

GW8510, 4- [ (7-oxo-6, 7-dihydro-8 h- [1, 3)]Thiazole [5,4-e ]]Indol-8-ylidene) methyl]Amino group]-n- (2-pyridyl) benzenesulfonamide, CAS: 222036-17-1, its molecular formula is C₂₁H₁₅N₅O₃S₂The chemical formula structure is:

is a yellow powder, has poor water solubility, can promote dissolution by heating, and has an average molecular weight of about 449.51 g/mol. In the present invention, GW8510 was dissolved in physiological saline at the time of administration to mice, and GW8510 was dissolved in a cell-complete medium in cell experiments.

Example 1GW8510 prolongs Yeast replication Life, delays cellular senescence and mitochondrial function

First, experiment method

1. Miniflow experiment for culturing budding yeast

In the aging field, higher animals or primate rhesus monkeys are the most ideal surrogate model for studying human aging, but there are many challenges to their study due to experimental complexity, long cycle length, high cost, and ethical reasons. However, for the organisms with low-grade short-life models, most of the organisms are relatively simple in system, low in cost and short in research time, and are ideal models for researching aging mechanisms. Therefore, from the practical point of view, it is extremely important to further develop the aging and the mechanism thereof by appropriately selecting the model organism related to aging and sufficiently exerting the advantages of the model organism. Saccharomyces cerevisiae is the simplest unicellular eukaryote in human aging research, the aging metabolic mechanism of the cell is similar to the human body cell metabolic mechanism, and the simple and unique growth metabolic rule makes the unicellular eukaryote become an important model organism for the cell aging research. The culture medium of budding yeast is divided into liquid culture medium SD and solid culture medium YEPD, which both contain amino acids and glucose required for yeast growth. Because the yeast has long survival time on the YEPD and has long survival time on the SD, the YEPD is selected for culturing the yeast and storing strains, and the SD is selected for carrying out a microflow experiment, thereby being beneficial to shortening the experiment period.

Firstly, a small amount of monoclonal colonies are picked from a YEPD culture medium and placed into an SD culture medium, shaking culture is carried out in a shaking table at the temperature of 30 ℃ for 20 hours, and then yeast growing to a certain amount is introduced into a microfluidic chip. And (3) shooting the yeast splitting condition inside the chip once every 10min by using microscopic shooting equipment, tracking and shooting for 48-60 h in total, and monitoring and recording the splitting times of the yeast in the whole life cycle so as to reflect whether the service life of the yeast is prolonged or not.

2. Cell recovery and cryopreservation

The frozen 2BS and WI-38 cells were taken out from the liquid nitrogen and immediately placed in a 37 ℃ water bath and shaken to be rapidly thawed. Transfer 2BS cells to centrifuge tubes (containing 5mL of complete culture) on a clean benchBasal), sealing with a sealing film, centrifuging at 800rpm for 4min, discarding the supernatant to obtain precipitate, and adding 1mL of complete culture medium. Gently blow the cells until the cells are dispersed into single cells, transfer the cells into a new sterile petri dish and place the dish at 37 ℃ with 5% CO₂Culturing in an incubator.

The cell sap obtained by digestion was transferred to a centrifuge tube, sealed with a sealing film and placed in a centrifuge at 800rpm for 4min, and the supernatant was discarded to collect the precipitate and added to 1mL of cell frozen stock solution (90% complete medium + 10% DMSO). Gently blowing and beating the cells, transferring the cells into a 1.5mL freezing tube, firstly placing the tube in a refrigerator at 4 ℃ for standing for 30min, then placing the tube in a refrigerator at-20 ℃ for standing for 2h, then placing the tube in a refrigerator at-80 ℃ for standing overnight, and finally freezing the tube in a liquid nitrogen tank every other day.

3. Cell culture and passage

The cells used in the in vitro experiments were cultured in the presence of CO in DMEM (2BS and MEM for WI 38) containing 10% fetal bovine serum, 100U/ml penicillin and 100mg/ml treptomycin₂In a cell culture box, the culture conditions are 37 ℃, saturated humidity and 5% CO ₂95% air.

When the cells grow to 80% -90%, pouring out the culture medium, slowly washing for 3 times by using PBS buffer solution, then adding 1mL of 0.125% trypsin, slightly shaking until the cells are completely covered, digesting for about 2min, immediately pouring out the pancreatin after the tentacles of the cell protrusions are observed to be retracted to be round under a microscope, and adding 1mL of complete culture medium to stop the digestion. And (3) gently and repeatedly blowing the cells by using a suction tube until the cells completely fall off, transferring the cells to a centrifuge tube, sealing the centrifuge tube by using a sealing film, centrifuging the cells for 4min at 800rpm, removing the supernatant, taking the precipitate, and adding 1mL of complete culture medium. Gently blow-beating until cells are dispersed into single cells, then subculturing into new sterile culture dishes at a ratio of 1:2 and placing at 37 ℃ with 5% CO₂Culturing in an incubator.

4. Cell counting

Taking cells to be counted, washing and digesting the cells in a super clean bench, adding a proper amount of complete cell culture medium, resuspending the cells, blowing the cells uniformly, sucking 10 mu L of cell suspension by using an 10/20 mu L pipette, slightly dropping the cell suspension to the edge of one side of a cover glass of a cell counting plate, filling the cell suspension between the cover glass and the counting plate, and placing the cell suspension 3Counting the number of the cells under an optical microscope by using a cell counter, and calculating according to a corresponding formula to obtain the final total number of the cells (the calculation formula: the total number of the cells/mL is four big cells/4 multiplied by 10)⁴one/mL)

5. Cell viability assay

The Cell Counting Kit-8 is a WST-8-based Cell viability detection Kit. WST-8 is a compound similar to MTT, and can be reduced by some dehydrogenase in mitochondria to generate orange formazan in the presence of an electron coupling reagent, after adding 10% CCK8 reagent to each well of a 96-well plate, incubating for 1h at 37 ℃, and measuring absorbance at 450nm by a microplate reader. Each group was set with 5 wells, and cell viability was calculated by the following formula:

Cell viability(％)＝(OD_treatment _group-OD_blank)/(OD_control _group-OD_blank)×100。

6. cell proliferation assay

Cells in logarithmic growth phase were trypsinized at 3.5X 10 per well³Each 100. mu.l of the cells was inoculated into a 96-well plate, each set was provided with 5 wells, and the cells were arranged in time-point order (

day

0,1,2,3,4,5, 6) from left to right, and each time-point was divided into a control group and an administration group. Edge wells of 96-well plates were filled with sterile PBS to prevent edge effects. Is placed in CO₂In a cell culture box, the culture conditions are 37 ℃, saturated humidity and 5% CO ₂95% air. After more than four hours after inoculation, when the cells adhere to the wall, the cell culture solution of the day 0 group is absorbed, 10 percent of CCK8 reagent is added into each hole, the incubation is carried out for 1 hour at 37 ℃, and the absorbance is measured by an enzyme-labeling instrument at 490 nm. And (4) changing the rest solutions, measuring the absorbance of the cells at the corresponding time point at the same time every day until monitoring for one week, and finally drawing a cell proliferation curve for one week according to the absorbance value.

7. Total RNA extraction

The culture medium was discarded and washed once with PBS. Every 10cm²Adding 1-2ml of RNAi sodium into the cells in the culture area, fully covering the cells on the surface, and then blowing the cells off by using a pipette; transferring into 1.5ml centrifuge tube, and repeatingBlowing, and standing on ice for 5 min; adding 1/5 volumes of chloroform, shaking vigorously for 15s, and standing at 4 deg.C for 5 min; centrifuging at 4 deg.C for 15min at 12,000 g; absorbing the upper water phase into a new centrifugal tube; adding isopropanol with the same volume, and reversing and uniformly mixing; standing at 4 deg.C for 10 min; centrifuging at 4 deg.C for 10min at 12,000 g; discarding the supernatant, and adding 1ml of 75% ethanol prepared with DEPC water; flicking the bottom of the tube to suspend the precipitate, and standing for 3-5 min; centrifuging at 4 deg.C for 5min at 12,000g, and discarding the supernatant; drying the precipitate for 2-5 min; adding appropriate amount of DEPC water to dissolve the precipitate. RNA integrity was checked by 1% agarose gel electrophoresis and the purity and concentration were determined by calculating OD260/OD 280.

8. Fluorescent quantitative PCR

RNA template, primers, 2 × UltraSYBR One Step RT-qPCRBuff (r With ROX), Superenzyme Mix and RNase-Free Water were dissolved and kept on ice until needed.

TABLE 1PCR reaction System 1

Vortex, shake, mix, centrifuge briefly, collect solution to the bottom of the tube. The thermal cycler was preheated to 45 ℃ and the PCR tubes were placed in the thermal cycler for reaction under the following reaction conditions.

TABLE 2PCR reaction conditions 2

9. Senescence-associated β -galactosidase (SA- β gal) staining (SABG staining)

In 1995, Dimri et al identified tissue senescence for the first time with senescence-associated β -galactosidase (SA- β Gal), and this method was widely used since, notably in 2009 by Wang et al, using a fine-comparison of the amount of SA- β Gal and DNA damage in the liver, to obtain comparable data information of 8% senescent cells in young mice and 17% senescent cells in old mice, the principle of the method was based on the specific, highly active expression of SA- β -Gal in senescent cells, while X-Gal in the stain was catalyzed by SA- β Gal to produce a blue product visible under a microscope, staining senescent cells without staining pre-senescent cells, resting cells, immortal cells or tumor cells.

The staining working solution is prepared according to the method of the specification of the cell aging β -galactosidase staining kit, is placed on a shaking table to shake for 30min, and is filtered by a 0.22 mu m microporous filter membrane after being taken out, the interference of staining solution crystallization on photo processing can be effectively improved by the method.

The operation method comprises the steps of absorbing and removing culture solution in a culture dish, slightly washing 3 times by PBS (PBS is added), carrying out 5min each time, adding quantitative β -galactosidase staining fixing solution (1 mL is added correspondingly to a six-hole plate, and 100 mu L is added correspondingly to a 96-hole plate), carrying out room temperature fixation for 15min, absorbing and removing cell fixing solution, washing cells for 3 times by PBS, absorbing and removing PBS 5min each time, adding quantitative staining working solution capable of completely covering the bottom of the culture dish (1 mL is added correspondingly to the six-hole plate, and 100 mu L is added correspondingly to the 96-hole plate), incubating overnight in a constant-temperature incubator (air) at 37 ℃, sealing the six-hole plate by a sealing film to prevent evaporation, and wrapping the six-hole plate by tinfoil to shield light.

The staining solution is discarded every other day, and a quantitative nuclear fixed red staining solution which can completely cover the bottom of the culture dish is added for counterstaining (the nuclear fixed red counterstaining makes the cell outline clearer), and the mixture is kept still for 7 min. And (4) removing the staining solution, adding PBS, observing under a microscope, and taking a 100-time picture for recording, wherein the positive cells stained with blue are the aged cells.

10. Mitochondrial membrane potential staining

Mitochondrial membrane potential staining kit was purchased from Saimer fly (cat # I34361). The cells grow to about 70 percent, serum-free culture medium is added for synchronization for 12 hours, and the administration growth is 24 hours; removing the cell growth medium; adding a cell staining solution to the cells; incubation at 37 ℃ for 30 min; washed with PBS and detected by confocal laser detection.

11. ATP content detection

The kit was purchased from Biyuntian (cat. s 0026). Sample preparation: the culture medium was aspirated, and 200. mu.l of lysis buffer was added to each well of 6-well plate to lyse the cells. When the cells were lysed, the cells were repeatedly aspirated using a pipette. After lysis, the cells were centrifuged at 12000g for 5 minutes at 4 ℃ and the supernatant was collected for subsequent measurement. Preparation of standard curve assay: and (3) thawing the reagent to be used on an ice bath, and diluting the ATP standard solution into an appropriate concentration gradient by using ATP detection lysate. Concentration gradients were set at 0.01, 0.03, 0.1, 0.3, 1,3 and 10 μ M. Preparing an ATP detection working solution: an appropriate amount of ATP detection working solution is prepared according to the proportion that 100 microlitres of ATP detection working solution is needed for each sample or standard. The reagents to be used were thawed on an ice bath. Taking a proper amount of ATP detection reagent, and diluting the ATP detection reagent by using ATP detection reagent diluent according to the proportion of 1: 9. The diluted ATP detection reagent is ATP detection working solution used for subsequent experiments. Measurement of ATP concentration: a. add 100. mu.l ATP detection working solution to the detection well or tube. The mixture was left at room temperature for 3-5 minutes to consume all background ATP, thereby reducing the background. b. Add 20. mu.l of sample or standard to the well or tube, mix it quickly with a gun (micropipette), and after at least 2 seconds, measure the RLU value with a chemiluminescence apparatus (luminometer).

Second, experimental results

1.GW8510 can prolong the life of budding yeast

Firstly, selecting classical senescence mode biological budding yeast as a research object, and detecting the service life of the budding yeast under the action of GW8510 with different concentrations by adopting a microflow technology. As a result, as shown in FIG. 1, the life of budding yeast in the administered group was changed to some extent as compared with the control group, and the life of budding yeast was significantly prolonged at a GW8510 concentration of 5. mu.M. Indicating that GW8510 improves the replicative senescence expression of budding yeast to some extent.

2. GW8510 can relieve cell replicative senescence

After preliminary validation of the anti-aging effects of GW8510 in model organism budding yeast, next two natural aging cell lines were used: 2BS (human embryonic lung diploid cells) and WI38 (human embryonic lung fibroblasts) are taken as research objects, and the anti-aging effect of GW8510 in vitro is further researched. Firstly, the influence of GW8510 on the cell viability of old people (PD45)2BS and WI38 is detected through a cell viability determination experiment. The results are shown in fig. 2A and B, GW8510 has no significant cytotoxic effect on 2BS and WI38 cells, and the cell viability is significantly higher than that of the control group. Then continuously exploring the influence of GW8510 on the cell proliferation capacity, tracking and detecting through a cell viability determination experiment and drawing a one-week proliferation curve, wherein the higher the absorbance value at 490nm indicates that the cell proliferation rate is higher, and the slow proliferation rate is also one of the important manifestations of cell aging. As shown in fig. 2C, GW8510 was able to significantly promote cell proliferation, while control cells grew with time, the proliferation rate slowed down, and the two sets of differences gradually increased with time.

3.GW8510 can improve cell replicative senescence

After the proliferative division ability of senescent cells was examined, WI-38 cells were continuously used as subjects to further investigate the effect of GW8510 on senescent cells, and cellular senescence SA- β -gal staining analysis was performed to observe changes in cell morphology, the results are shown in fig. 3A, where senescent WI-38(PD45) stained more positively than young WI-38(PD30) cells β -galactosidase, suggesting cellular senescence, whereas after administration of GW8510, the positive rate of staining with fibroblast β -galactosidase was significantly decreased compared to the control group, followed by examination of the expression of p21, which is a senescence-associated marker gene in young senescent cells, the results are shown in fig. 3B, where p21 expression is significantly increased in senescent cells, and significantly decreased after administration of stem cells.

4.GW8510 can improve mitochondrial function and up-regulate expression of cycle-associated genes

Next, whether GW8510 can improve mitochondrial function was examined, and it was found that mitochondrial membrane potential significantly increased with intervention of GW8510, and it was statistically significant, as shown in fig. 4A-B, and that there was also a significant difference in the amount of ATP produced, and the above results indicate that GW8510 can improve mitochondrial function. Finally, GW8510 was also found to upregulate the expression of cycle-associated genes, as shown in FIG. 4C.

Example 2

First, experiment method

1.GW8510 can delay the life of the mice with natural aging

The aging resistance of GW8510 to naturally aging mice was investigated using 18-month-old naturally aging ICR mice as subjects. The aged mice of 18 months of age were injected with physiological saline, 2mg/kg and 10mg/kg (the administration dose of human was 0.22mg/kg and 1.1mg/kg in comparison with the administration dose of mouse and human), once a day, continuously administered for one week, stopped for 3 weeks, repeated for 2 times, and the survival time of the mice was recorded during the administration period, and the results are shown in fig. 5, and the life of the mice was significantly prolonged under the intervention of GW 8510.

2. GW8510 can improve the healthy life span of naturally aging mice

The method is characterized in that a 16-month-old natural aging C57 mouse is taken as a target, the 16-month-old aged mouse is injected with normal saline, 2mg/kg and 10mg/kg respectively, the administration is performed once a day, the administration is continued for one week, the administration is stopped for 3 weeks, the administration is repeated for 4 times, the interval is 2 months, the administration is performed once a day, the administration is continued for one week, the administration is stopped for 3 weeks, and the administration is repeated for 2 times, and under the condition of administration, the cognitive ability (shown in figures 6A and D), the muscle ability (shown in figure 6B) and the motor ability (shown in figure 6C) of the.

3.GW8510 acts on PAK1 protein

First, a possible target was searched for in yeast, and it was found that the effect of prolonging the life of GW8510 still existed in yck1 knock-out strain (see FIG. 7A), ypk2 knock-out strain (see FIG. 7B), tpk2 knock-out strain (see FIG. 7C), cap1 knock-out strain (see FIG. 7D). In the ste20 (see fig. 7E) knockout strain, GW8510 did not prolong its life, so it was mapped to PAK1 protein, which is a human protein, and it was found that GW8510 binds PAK1 protein with a binding coefficient kd (m) of 8.155E-07 (see fig. 7E).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of GW8510 for the manufacture of a medicament for prolonging the lifespan, improving cognitive ability and/or improving muscle ability and/or improving motor ability of a mammal in its natural aging.
2. Use according to claim 1, characterized in that: the administration dose of GW8510 is 0.22 mg/kg/day-1.1 mg/kg/day.
The application of GW8510 in preparing drugs for inhibiting the expression of cell cycle related protein p21 and/or CDK family genes.
Use of GW8510 for the manufacture of a medicament for the treatment and/or amelioration of mitochondrial function of senescent cells.
Application of GW8510 in preparing medicine for prolonging life of replicative senescence cell line.
6. Use according to claim 5, characterized in that: the senescent cell line comprises 2BS, WI 38.