WO2021179952A1

WO2021179952A1 - Application of gw8510 in preparation of drugs for prolonging lives, improving cognitive ability, and the like of mammals in natural aging

Info

Publication number: WO2021179952A1
Application number: PCT/CN2021/078628
Authority: WO
Inventors: 谢正伟; 安永盼; 杨宝学; 朱杰
Original assignee: 北京大学
Priority date: 2020-03-09
Filing date: 2021-03-02
Publication date: 2021-09-16
Also published as: CN111228266A; CN111228266B

Abstract

Provided is an application of GW8510 in preparation of drugs for prolonging lives, and improving cognitive ability and/or muscle ability and/or motion ability of mammals in natural aging. Research on an in-vitro mechanism shows that GW8510 can regulate expressions of a cell cycle-related protein p21 and a CDK family gene, thereby improving a replicative senescence state of natural aging cell lines 2BS and WI38, and improving a mitochondrial function of aging cells. A mouse experiment shows that GW8510 can prolong the life of a mouse in normal aging.

Description

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

Technical field

The invention belongs to the field of biology, and particularly relates to the application of GW8510 in the preparation of medicines for prolonging life span and improving cognition during natural aging of mammals.

Background technique

Aging is a process in which the function of tissues and organs in the body degenerate with age, and is closely related to normal embryonic development, body aging, and aging-related diseases. In recent years, the research on cell aging has become more and more in-depth, and its biological role in anti-aging has also been paid more and more attention. Studies have shown that cellular senescence refers to the gradual decline of the normal physiological functions and proliferation ability of cells over time or when faced with external stress and pressure, so as to break away from the cell cycle. This process is related to tumor, tissue regeneration and The aging of the body has important links. Since the theory of mitochondrial aging was put forward, mitochondria have become the focus of aging research. With age, the function of mitochondria will gradually weaken, and at the same time, with age, cell degradation is caused by reactive oxygen species (ROS). The mitochondrial aging theory regards mitochondria as the main producer of ROS. At present, there are still few studies on GW8510. Existing studies have shown that GW8510 can reduce the side effects of chemotherapy, prevent hair loss and mucositis.

Summary of the invention

In order to extend the life span of mammals when they naturally age, the present invention provides the application of GW8510.

The first aspect of the present invention provides the application of GW8510 in the preparation of drugs for prolonging lifespan, improving cognitive ability and/or improving muscle ability and/or improving exercise ability of mammals during natural aging.

Preferably, the dosage of GW8510 is 0.22 mg/kg/day to 1.1 mg/kg/day.

The second aspect of the present invention provides the application of GW8510 in the preparation of drugs for inhibiting the expression of cell cycle-related protein p21 and/or CDK family genes.

The third aspect of the present invention provides the application of GW8510 in the preparation of drugs for treating and/or improving mitochondrial function of senescent cells.

The fourth aspect of the present invention provides the application of GW8510 in the preparation of drugs for prolonging the lifespan of replicative senescent cell lines.

Preferably, the senescent cell line includes 2BS and WI38.

Beneficial effects: The present invention provides that GW8510 can significantly improve the healthy aging of naturally aging mice and prolong their lifespan. According to in vitro mechanism studies, GW8510 can regulate the expression of cell cycle-related protein p21 and CDK family genes, thereby improving the natural aging cell line 2BS And the replicative senescence state of WI38. It can also improve the mitochondrial function of senescent cells, thereby slowing down aging.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it in accordance with the content of the description, the preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

Description of the drawings

Figure 1 shows that GW8510 can extend the life of budding yeast.

Figure 2 shows that GW8510 can alleviate the replicative senescence of 2BS (PD45) and WI38 (PD45) cells; among them, GW8510 can analyze the viability of aged 2BS (A) and WI38 (B) cells. The effect of 2BS(C) cell proliferation in PD45. Mean±SEM.n=5-8. *P<0.05; **P<0.01; ***P<0.001.

Figure 3 shows that GW8510 can improve the cell senescence morphology of elderly WI-38; among them, (A) cell senescence SA-β-gal staining (magnification × 400); (B) cell senescence marker p21 gene expression change after administration; (C) Changes in the expression of cellular senescence-associated secreted phenotype (SASP) mRNA after administration. Mean±SEM.n=5-8. *P<0.05; **P<0.01; ***P<0.001.

Figure 4 shows that GW8510 can affect cell mitochondrial function and the expression of cycle-related genes; among them, (A) cell mitochondrial membrane potential staining (TMRM); (B) mitochondrial membrane potential staining statistics; (C) cell ATP production; (D) ) Changes in mRNA expression of cell cycle-related genes. Mean±SEM.n=5-8. *P<0.05; **P<0.01; ***P<0.001.

Figure 5 shows that GW8510 can prolong the lifespan of naturally aging mice; using ICR mice, drug intervention was started at the age of 18 months.

Figure 6 shows that GW8510 can prolong the healthy lifespan of naturally aging mice; among them, C57 mice were used to start the drug at 16 months (A) Novel object recognition experiment (characterizing cognitive ability); (B) Grip strength experiment (characterizing muscle ability) ; (C) Fatigue Rotary Rod Test (to characterize sports ability); (D) Y-maze experiment (to characterize cognitive ability).

Figure 7 shows that the target of GW8510 is PAK1; among them, (AD) yeast gene knockout strain GW8510 interferes with the life curve; (E) ste20 (PAK1 yeast homology) knockout strain life curve; (F) GW8510 and PAK1 protein SPR (Surface Plasmon Resonance) experiment.

Detailed ways

The specific implementation of the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention.

ICR mice, male, 8 weeks, 20-22g, were purchased from the Experimental Animal Center of Peking University Health Science Center.

Maintenance feed: purchased from the Laboratory Animal Center of Peking University Health Science Center.

C57BL/6J mice, male, 13 months old, 25-28g, purchased from Beijing Sibeifu Experimental Animal Center.

All animal experiments are strictly in accordance with the requirements of the "Guidelines for the Management and Use of Laboratory Animals", and have been approved by the Peking University Laboratory Animal Management Committee. In the process of killing the animal, under anesthesia and try to reduce the animal's suffering.

2BS cells (human diploid fibroblasts) and WI-38 cells (human embryo lung fibroblasts) were purchased from the China Institute for Food and Drug Control.

GW8510,4-[(7-oxo-6,7-dihydro-8h-[1,3]thiazole[5,4-e]indole-8-ylidene)methyl]amino]-n-( 2-pyridyl)benzenesulfonamide, CAS: 222036-17-1, its molecular formula is C ₂₁ H ₁₅ N ₅ O ₃ S ₂ , and the chemical formula structure is:

It is a yellow powder with poor water solubility and can be dissolved by heating. The average molecular weight is about 449.51g/mol. In the present invention, GW8510 is dissolved in physiological saline during administration to mice, and GW8510 is dissolved in complete cell culture medium in cell experiments.

Example 1 GW8510 prolongs yeast replication life, delays cell senescence and mitochondrial function

1. Experimental method

1. Microfluidic experiment of budding yeast culture

In the field of aging, higher animals or primate rhesus monkeys are the most ideal alternative models for studying human aging. However, due to the complexity of the experiment, the long cycle, the high cost, and many ethical reasons, there are many challenges to their research. . But for low-level short-lived model organisms, most of them have relatively simple systems, low cost, and short research time, making them ideal models for studying the mechanism of aging. Therefore, starting from practical problems, properly selecting aging-related model organisms and giving full play to the advantages of model organisms is extremely important to further improve the research on aging and its mechanisms. Saccharomyces cerevisiae is the simplest single-celled eukaryote in the study of human aging. Its cellular aging metabolism mechanism is similar to that of human cells. Its simple and unique growth and metabolism law makes it an important model organism for the study of cell aging. The culture medium of budding yeast is divided into liquid medium SD and solid medium YEPD, both of which contain amino acids and glucose required for yeast growth. Because the survival time of yeast on YEPD is much longer than that of SD, YEPD is selected for yeast cultivation and strain preservation, and SD is used for microfluidic experiments, which is beneficial to shorten the experimental period.

First pick a small number of monoclonal colonies from the YEPD medium and put them into the SD medium, culture them in a shaker at 30°C for 20 hours, and then pass the yeast that has grown to a certain amount into the microfluidic chip. Using a photomicrograph device, the internal yeast splitting of the chip is photographed every 10 minutes, a total of 48-60h is tracked and photographed, and the number of splits of the yeast during the entire life cycle is monitored and recorded to reflect whether the lifespan of the yeast has been extended.

2. Cell recovery and cryopreservation

Remove the cryopreserved 2BS and WI-38 cells from the liquid nitrogen, and immediately put them in a 37°C water bath and shake them to quickly melt them. Transfer the 2BS cells to a centrifuge tube (containing 5 mL of complete medium) on an ultra-clean workbench, seal the parafilm and place it in a centrifuge at 800 rpm for 4 minutes, discard the supernatant and collect the precipitate, and then add 1 mL of complete medium. After gently pipetting until the cells are dispersed and single, they are transferred to a new sterile petri dish and placed in a 37°C 5% CO ₂ incubator for culture.

The digested cell liquid was transferred to a centrifuge tube, and the sealing film was sealed and placed in a centrifuge at 800 rpm for 4 min centrifugation. The supernatant was discarded and the precipitate was added to 1 mL of cell cryopreservation solution (90% complete medium + 10% DMSO). Gently pipette the cells and transfer them to a 1.5mL cryopreservation tube. Place them in a refrigerator at 4°C for 30 minutes, then place in a refrigerator at -20°C for 2 hours, then in a refrigerator at -80°C overnight, and freeze in the solution the next day. Nitrogen tank.

3. Cell culture and passage

The cells used in the in vitro experiment use DMEM (MEM for 2BS and WI38) culture medium containing 10% fetal bovine serum, 100U/ml penicillin and 100mg/ml streptomycin, placed in a CO ₂ cell incubator, and the culture condition is 37°C. Saturated humidity, 5% CO ₂ , 95% air.

When the cells grow to 80%-90%, discard the medium, rinse slowly with PBS buffer 3 times, then add 1 mL of 0.125% trypsin, shake gently to completely cover the cells, digest for about 2 minutes, and observe under a microscope After the tentacles of the cell protrusions retracted into a round shape, the trypsin was immediately discarded, and 1 mL of complete medium was added to terminate the digestion. Use a pipette to gently blow the cells repeatedly until all of them fall off, transfer to a centrifuge tube, seal with a parafilm and place it in a centrifuge at 800 rpm for 4 min. Discard the supernatant and leave the precipitate to add 1 mL of complete medium. After gently pipetting until the cells are dispersed and single, pass them to a new sterile petri dish at a ratio of 1:2 and place them in a 37°C 5% CO ₂ incubator for culture.

4. Cell count

Take the cells to be counted, rinse and digest them in an ultra-clean table, add an appropriate amount of cell complete medium, resuspend the cells and pipette evenly, use a 10/20μL pipette to suck up 10μL of cell suspension, and gently drop it onto the cover glass of the cell counting plate Fill the cell suspension between the cover glass and the counting plate on the edge of one side of the slide, and place it for 3 minutes (note that there are no bubbles under the cover glass to avoid affecting the accuracy of counting), and count with a cell counter under an optical microscope. Then calculate the total number of cells according to the corresponding formula (calculation formula: total number of cells/mL = total number of four large cells/4×10 ⁴ cells/mL)

5. Cell viability determination

Cell Counting Kit-8 is a cell viability detection kit based on WST-8. WST-8 is a compound similar to MTT. In the presence of electronic coupling reagents, it can be reduced by some dehydrogenases in the mitochondria to form orange-yellow formazan. After adding 10% CCK8 reagent to each well of a 96-well plate, 37 Incubate for 1h at ℃, measure the absorbance with a microplate reader at 450nm. Each group has 5 multiple wells, and the cell viability is calculated by the following formula:

Cell viability(%)=(OD _{treatment group} -OD _blank )/(OD _{control group} -OD _blank )×100.

6. Cell proliferation detection

The cells in the logarithmic growth phase were digested with trypsin, and ^{seeded in a 96-well plate at a density of 3.5×10 3} cells/100μl per well. There were 5 multiple wells in each group. 1,2,3,4,5,6 days), each time point is divided into a control group and an administration group. The edge holes of the 96-well plate are filled with sterile PBS to prevent edge effects. Placed in a CO ₂ cell incubator, culture conditions are 37° C., saturated humidity, 5% CO ₂ , 95% air. Allow the cells to adhere to the wall more than four hours after inoculation, aspirate the cell culture medium of the 0th day group, add 10% CCK8 reagent to each well, incubate at 37°C for 1 hour, measure the absorbance with a microplate reader at 490nm. The rest were changed separately, and then the cell absorbance at the corresponding time point was measured at the same time every day until one week of monitoring, and finally the cell proliferation curve was drawn for one week according to the absorbance value.

7. Total RNA extraction

Discard the culture medium and wash once with PBS. Add 1-2ml RNA isolater per 10cm ² culture area of cells to fully cover the cell surface, then pipette the cells down; move to a 1.5ml centrifuge tube, pipette repeatedly, and let stand on ice for 5min; add 1/ 5 volumes of chloroform, vigorously shake for 15s, 4℃ for 5min; 12,000g, 4℃, centrifugation for 15min; pipette the upper aqueous phase into a new centrifuge tube; add equal volume of isopropanol, invert and mix; 4℃ 10min; centrifuge at 12,000g for 10min at 4℃; discard the supernatant and add 1ml of 75% ethanol prepared with DEPC water; flick the bottom of the tube to suspend the precipitate and let it stand for 3-5min; centrifuge at 12,000g at 4℃ for 5min and discard Clear; dry the precipitation for 2-5 minutes; add an appropriate amount of DEPC water to dissolve the precipitation. Use 1% agarose gel electrophoresis to detect RNA integrity, and calculate OD 260/OD 280 to detect purity and concentration.

8. Fluorescence quantitative PCR

Dissolve the RNA template, primers, 2×UltraSYBR One Step RT-qPCR Buffe(r With ROX), SuperEnzyme Mix and RNase-Free Water and place them on ice for later use.

Table 1 PCR reaction system 1

Vortex to mix well, centrifuge briefly, and collect the solution at the bottom of the tube. Preheat the thermal cycler to 45°C, place the PCR tube in the thermal cycler, and perform the reaction under the following reaction conditions.

Table 2 PCR reaction conditions 2

9. Aging-related β-galactosidase (SA-βgal) staining (SABG staining method)

In 1995, Dimri et al. used the aging-related β-galactosidase (SA-βgal) to identify tissue aging for the first time. Since then, this method has been widely used. The more famous one is that Wang et al. used SA-βgal in the liver in 2009. Comparing with the amount of DNA damage, comparable data can be obtained: 8% of senescent cells in young mice and 17% of senescent cells in old mice. The principle of this method is based on the specific and highly active expression of SA-β-gal in senescent cells, and X-Gal in the dye is catalyzed by SA-βgal to produce a blue product visible under the microscope, thereby staining senescent cells without It stains pre-senescent cells, quiescent cells, immortal cells or tumor cells.

The staining working solution is configured according to the instructions of the "Cell Senescence β-Galactosidase Staining Kit", and then placed on a shaker for 30 minutes. Interference in photo processing. The working solution of the dyeing solution should be protected from light, and it should be prepared immediately.

Operation method: aspirate the culture medium in the petri dish, rinse gently with PBS 3 times, 5 min each time, and add quantitative β-galactosidase staining fixative (1mL for a six-well plate and 100μL for a 96-well plate), Fix for 15 min at room temperature, aspirate the cell fixation solution, and wash the cells 3 times with PBS for 5 min each time. Aspirate PBS, and add quantitative staining working solution that can completely cover the bottom of the petri dish to each well (1mL for a six-well plate and 100μL for a 96-well plate). Incubate overnight in a 37°C constant temperature incubator (air), seal the six-well plate with a parafilm to prevent evaporation, and then wrap the six-well plate with tin foil to shade the plate.

The next day, discard the staining solution, add a quantitative nuclear fast red staining solution that can completely cover the bottom of the petri dish for counterstaining (nuclear fast red counterstaining makes the cell contour clearer), and let it stand for 7 minutes. Discard the staining solution, add PBS, observe under a microscope, take a photo at 100 times and record. The positive cells stained with blue are senescent cells.

10. Mitochondrial membrane potential staining

Mitochondrial membrane potential staining kit was purchased from Thermo Fisher Scientific (Cat. No.: I34361). The cells grow to about 70%, add serum-free medium for synchronization for 12 hours, and grow for 24 hours after administration; remove the cell growth medium; add cell staining solution to the cells; incubate at 37°C for 30 minutes; wash with PBS and use laser confocal Detection.

11. ATP content detection

The kit was purchased from Biyuntian Company (Cat. No.: s0026). Sample preparation: Aspirate the culture medium, add 200 microliters of lysate to each well of the 6-well plate to lyse the cells. In order to lyse the cells sufficiently, use a pipette to repeatedly pipette. After lysis, centrifuge at 12000g at 4°C for 5 minutes, and take the supernatant for subsequent determination. Preparation for standard curve determination: melt the reagents to be used on an ice bath, and dilute the ATP standard solution with ATP detection lysate to an appropriate concentration gradient. Set the concentration gradient 0.01, 0.03, 0.1, 0.3, 1, 3 and 10μM. Preparation of ATP detection working solution: Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 microliters of ATP detection working solution for each sample or standard. Dissolve the reagents to be used on an ice bath. Take an appropriate amount of ATP detection reagent and dilute the ATP detection reagent with the ATP detection reagent diluent in a ratio of 1:9. The diluted ATP detection reagent is the ATP detection working solution for subsequent experiments. Determination of ATP concentration: a. Add 100 microliters of ATP detection working solution to the detection hole or detection tube. Leave it at room temperature for 3-5 minutes, so that all the background ATP is consumed, thereby reducing the background. b. Add 20 microliters of sample or standard to the test hole or test tube, and quickly mix it with a gun (micropipette). After an interval of at least 2 seconds, use a luminometer to determine the RLU value.

2. Experimental results

1. GW8510 can extend the life of budding yeast

First, the classical aging model biological budding yeast was selected as the research object, and the lifespan of budding yeast under the action of different concentrations of GW8510 was detected by using microfluidic technology. The results are shown in Figure 1. Compared with the control group, the lifespan of budding yeast in the administration group has changed to a certain extent, and when the concentration of GW8510 is 5μM, the lifespan of budding yeast can be significantly prolonged. It shows that GW8510 improves the replicative senescence performance of budding yeast to a certain extent.

2. GW8510 can alleviate cell replicative senescence

After preliminary verification of the anti-aging effect of GW8510 in the model organism budding yeast, two natural senescent cell lines: 2BS (human embryonic lung diploid cells) and WI38 (human embryo lung fibroblasts) were used as research Subject, to further explore the anti-aging effect of GW8510 in vitro. Firstly, the cell viability assay experiment was used to detect the effect of GW8510 on the viability of elderly (PD45) 2BS and WI38 cells. The results are shown in Figure 2A and B. GW8510 has no obvious cytotoxic effect on 2BS and WI38 cells, and the cell viability is significantly higher than that of the control group. Then continue to explore the effect of GW8510 on cell proliferation. Through cell viability assay experiments, we will track and draw a week of proliferation curve. The larger the absorbance at 490nm wavelength, the higher the cell proliferation rate, and the slower proliferation rate is also an important manifestation of cell aging. one. The results are shown in Figure 2C, GW8510 can significantly promote cell proliferation, while the control group cells increase with time, the proliferation rate slows down, and the difference between the two groups gradually increases with time.

3. GW8510 can improve cell replicative senescence

After testing the ability of senescent cells to proliferate and divide, continue to use WI-38 cells as the research object to further explore the effect of GW8510 on senescent cells. Cell senescence SA-β-gal staining analysis was performed to observe the changes in cell morphology. The results are shown in Figure 3A. Compared with young WI-38 (PD30) cells, the old WI-38 (PD45) stained more positively for β-galactosidase, suggesting cell senescence. Compared with the control group, the senescence staining results after administration of GW8510 decreased significantly. Then, the expression of p21, a marker gene associated with senescence, was detected in young and senescent cells. The results are shown in Figure 3B, the expression of p21 in senescent cells was significantly increased, but it was significantly decreased after administration intervention. Finally, the changes in the expression of secreted phenotype genes related to cellular senescence after administration were detected. The results are shown in Figure 3C. Compared with the control group, the expression levels of these marker genes were significantly down-regulated in the administration group, and there were statistical differences. The above results suggest that GW8510 has certain anti-aging effects from the staining of senescent cells and changes in senescence-related gene expression.

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

Then we tested whether GW8510 can improve mitochondrial function. It was found that under the intervention of GW8510, the mitochondrial membrane potential increased significantly, and it was statistically significant, as shown in Figure 4A-B. At the same time, the amount of ATP production was also detected, and there was also a significant difference. The above results indicate that GW8510 can improve mitochondrial function. Finally, it was found that GW8510 can up-regulate the expression of cycle-related genes, as shown in Figure 4C.

Example 2

1. Experimental method

1. GW8510 can delay the lifespan of naturally aging mice

Using 18-month-old naturally aging ICR mice as the object, explore the anti-aging effect of GW8510 on naturally aging mice. 18-month-old aging mice were injected with physiological saline, 2mg/kg and 10mg/kg (according to the comparison of the dose relationship between mice and humans, the human dose is 0.22mg/kg, 1.1mg/kg), daily Once, the administration was continued for one week, stopped for 3 weeks, and repeated twice. During the administration, the survival time of the mice was recorded. The results are shown in Figure 5. The life span of the mice was significantly prolonged under the intervention of GW8510.

2. GW8510 can improve the healthy lifespan of naturally aging mice

Using 16-month-old naturally aging C57 mice as the object, the 16-month-old aging mice were injected with physiological saline, 2mg/kg and 10mg/kg, once a day for one week, stopping for 3 weeks, repeating 4 times, intermittently For 2 months, do it once a day, continue the administration for one week, stop for 3 weeks, and repeat twice. Under the condition of administration, the mice’s cognitive ability (Figure 6A, D), muscle ability (Figure 6B), exercise Ability (as shown in Figure 6C) has been significantly improved.

3. GW8510 acts on PAK1 protein

First, we searched for possible targets in yeast, and found that yck1 gene knockout strains (Figure 7A), ypk2 gene knockout strains (Figure 7B), tpk2 gene knockout strains (Figure 7C), cap1 gene knockout strains (As shown in Figure 7D), the life extension effect of GW8510 still exists. In the ste20 (Figure 7E) knockout strain, GW8510 cannot extend its lifespan, so it corresponds to the human protein PAK1 protein, and it is found that GW8510 can bind to PAK1 protein, the binding coefficient KD(M)=8.155E- 07 (Figure 7E).

The above-mentioned embodiments only express several implementation modes of the present invention, and their description is relatively specific and detailed, but they should not be understood as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

The application of GW8510 in the preparation of medicines for extending life expectancy, improving cognitive ability and/or improving muscle ability and/or improving exercise ability of mammals during natural aging.
The application according to claim 1, wherein the dosage of GW8510 is 0.22 mg/kg/day to 1.1 mg/kg/day.
Application of GW8510 in the preparation of drugs for inhibiting the expression of cell cycle-related protein p21 and/or CDK family genes.
Application of GW8510 in the preparation of medicines for treating and/or improving mitochondrial function of senescent cells.
Application of GW8510 in the preparation of drugs for prolonging the life span of replicative senescent cell lines.
The application according to claim 5, wherein the senescent cell line includes 2BS and WI38.