CN115671089B - Application of gallic acid in delaying cell aging - Google Patents
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- CN115671089B CN115671089B CN202210882952.5A CN202210882952A CN115671089B CN 115671089 B CN115671089 B CN 115671089B CN 202210882952 A CN202210882952 A CN 202210882952A CN 115671089 B CN115671089 B CN 115671089B
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Abstract
The invention discloses application of gallic acid in delaying cell senescence. The invention discovers that gallic acid has the effect of delaying senescence, can delay senescence of stem cells with premature senility, and comprises improvement of self-renewal capacity of the stem cells, reduction of protein levels of senescence markers P16 and P21, improvement of multipotent stem of the stem cells, improvement of genome stability and mitochondrial function and the like. Gallic acid is described as an "anti-aging agent (geroprotector)" drug. This further expands the current anti-aging compounds, providing a very promising candidate strategy for anti-aging, which will help to alleviate the increasingly more aging problems.
Description
Technical Field
The invention relates to application of gallic acid in delaying cell aging in the field of biological medicine.
Background
In recent years, the aging problem of the world population is becoming more and more advanced, and the aging is often one of the main risk factors for a number of chronic diseases, such as senile dementia, osteoarthritis, cardiovascular diseases, cancer, etc. The increase of the population of the elderly and the incidence rate of diseases can cause great impact on the support system and the medical system of the aged in various countries, and seriously affect the daily life of people. Although aging is unavoidable, it is regulatable. Thus, delaying aging, i.e., improving health life or survival, and preventing aging-related diseases would be the best solution to the aging challenges. The current aging research has become a major hotspot in the life field, and various means for delaying aging have also been developed, such as small molecule drug intervention, lifestyle intervention (exercise, diet restriction), gene therapy, and the like. Of these, the potential for use of small molecule drugs to delay aging is optimal. Through research in many models of organisms, including yeasts, nematodes, drosophila and mice, many compounds have now been discovered that can delay the onset or progression of aging, which are multi-targeted to nutrient signaling pathways, oxidative stress, endogenous metabolite supplementation, etc., such as rapamycin, vitamin C, quercetin, nicotinamide, etc. However, the safety and efficacy of most compounds remains to be evaluated in a deep, versatile manner, particularly in clinical trials. It is known that only a few drugs are currently approved for clinical trials, including metformin, nicotinamide, but the results of the trial do not meet one's expectations: improving the survival of the subject. Therefore, it is crucial to identify more and effective anti-aging drugs, and this will have a very positive driving effect on alleviating the aging crisis of the population.
Gallic acid is a natural phenolic compound which is mostly present in tea, fruit and wine, has wide biological effects and relates to biological functions such as anticancer, antibacterial, anti-inflammatory and antiviral activities.
Disclosure of Invention
The technical problem to be solved by the invention is how to inhibit the aging of aging stem cells.
In order to solve the technical problems, the invention firstly provides application of gallic acid in preparing a product with any one of the following functions:
X1, inhibiting cellular senescence;
X2, regulating and controlling the cell cycle;
x3, slowing down the rate of cell degeneration or enhancing the in vivo retention capacity of cells;
X4, slowing down cell telomere shortening;
X5, regulating cellular mitochondrial homeostasis;
x6, improving the capability of the cells to resist external stimulus;
X7, treating and/or preventing aging in an animal;
X8, improving animal muscle strength;
X9, improving the exercise capacity of animals;
x10, improving the memory capacity of animals.
In the above application, the cells may be senescent cells. The cells may be stem cells (e.g., mesenchymal stem cells) or endothelial cells (e.g., aortic endothelial cells).
Further, the cells may be senescent stem cells or senescent endothelial cells. The senescent stem cells may be senescent mesenchymal stem cells and the senescent endothelial cells may be senescent human aortic endothelial cells.
The senescent stem cells can be further senescent mesenchymal stem cells of targeted knockout WRN, or senescent mesenchymal stem cells of LMNA gene mutation, or replicative senescent mesenchymal stem cells caused by continuous passage, or senescent mesenchymal stem cells induced by UV or H 2O2. The senescent mesenchymal stem cells can be differentiated from human embryonic stem cells (such as human embryonic stem cell H9 cell line).
The senescent endothelial cells may be replicative senescent endothelial cells.
In such applications, the modulation of cell cycle may be manifested as a reversal of aging stem cell growth arrest.
The modulation of cellular mitochondrial homeostasis may be manifested at a level that reduces the level of ROS in the mitochondria, or that upregulates mitochondrial membrane potential, or that reduces mitochondrial mass.
The external stimulus is a stimulus capable of triggering cell aging, such as ultraviolet light (UV) or hydrogen peroxide (H 2O2).
In the above application, the animal aging may be premature animal aging. The animal presenility can be mammal presenility, such as human presenility. The animal aging may be manifested in an increase in muscle strength, an increase in motor capacity, and/or an increase in memory capacity of the animal.
The invention also provides application of the gallic acid in preparation of the stem cell stem product for maintaining aging.
In the above application, the senescent stem cells may be senescent mesenchymal stem cells.
The senescent stem cells can be further senescent mesenchymal stem cells of targeted knockout WRN, or senescent mesenchymal stem cells of LMNA gene mutation, or replicative senescent mesenchymal stem cells caused by continuous passage, or senescent mesenchymal stem cells induced by UV or H 2O2. The senescent mesenchymal stem cells can be differentiated from human embryonic stem cells (such as human embryonic stem cell H9 cell line).
In the above application, the maintenance of stem cell stem property may be represented by activating the differentiation potential of stem cells to adipocytes, activating the differentiation potential of stem cells to osteoblasts, or activating the differentiation potential of stem cells to chondrocytes.
The invention also provides application of the gallic acid in preparing a product for promoting proliferation of the aging stem cells.
In the above application, the senescent stem cells may be senescent mesenchymal stem cells.
The senescent stem cells can be further senescent mesenchymal stem cells of targeted knockout WRN, or senescent mesenchymal stem cells of LMNA gene mutation, or replicative senescent mesenchymal stem cells caused by continuous passage, or senescent mesenchymal stem cells induced by UV or H 2O2. The senescent mesenchymal stem cells can be differentiated from human embryonic stem cells (such as human embryonic stem cell H9 cell line).
The invention discovers that gallic acid (3, 4, 5-trihydroxybenzoic acid, GALLIC ACID, GA) has the effect of delaying senescence, can delay senescence of stem cells with premature senility, and comprises the steps of improving self-renewal capacity of the stem cells, reducing protein levels of senescence markers P16 and P21, improving multipotent stem cells, stabilizing genome, improving mitochondrial function and the like. Gallic acid is described as an "anti-aging agent (geroprotector)" drug. This further expands the current anti-aging compounds, providing a very promising candidate strategy for anti-aging, which will help to alleviate the increasingly more aging problems.
Drawings
FIG. 1 is a graph showing the results of a test for gallic acid improving the senescence phenotype and self-renewal capacity of WS hMSC; wherein A is a cell growth curve contrast analysis chart; b is an analysis chart of monoclonal formation experimental results; c is an analysis chart of SA-beta-Gal staining results; d is a Western blot detection analysis chart of P16, P21, LAP2 beta and Lamin B1; e is a detection result analysis chart of the cell cycle; f is a statistical graph of telomere length results. The control in the figure represents WS hMSC after dimethyl sulfoxide treatment, and gallic acid represents WS hMSC after 1. Mu.M gallic acid treatment; * P <0.05 and P <0.01, P <0.001. Scale bar 100 μm.
FIG. 2 is a graph showing the results of a test for enhancing the pluripotent dry function of WS hMSC with gallic acid; wherein A is an in vivo persistence capacity detection result analysis chart; b is an analysis chart of the result of WS hMSC differentiation to the three lines. The control in the figure represents WS hMSC after dimethyl sulfoxide treatment, and gallic acid represents WS hMSC after 1. Mu.M gallic acid treatment; * P <0.05 and P <0.01, P <0.001. Scale bar 100 μm.
FIG. 3 is a graph showing the results of a gallic acid-mediated WS hMSC mitochondrial homeostasis test; wherein A is a detection result diagram of mitochondrial active oxygen content; b is a mitochondrial quality level detection analysis chart; c is a mitochondrial membrane potential level detection analysis graph. The control in the figure represents WS hMSC after dimethyl sulfoxide treatment, and gallic acid represents WS hMSC after 1. Mu.M gallic acid treatment; * P <0.05 and P <0.01, P <0.001.
FIG. 4 is a graph showing the results of experiments on gallic acid to alleviate various aging cell models; wherein A is an analysis chart of experimental results formed by monoclonal in an aging mesenchymal stem cell model; b is an analysis chart of SA-beta-Gal staining results in an aging mesenchymal stem cell model; c is an analysis chart of SA-beta-Gal staining results in human aortic endothelial cells. In the figure, the control represents cells after dimethyl sulfoxide treatment, and gallic acid represents cells after 1. Mu.M gallic acid treatment; * P <0.05 and P <0.01, P <0.001. Scale bar 100 μm.
FIG. 5 is a test result of a transcriptome analysis of WS hMSC and HGPS hMSC treated with gallic acid; wherein A is the pathway display of differential gene enrichment; b is the expression of senescence-associated genes.
FIG. 6 shows that gallic acid can delay the senescence-associated phenotype of normal mice. A is a holding power measurement experiment result, B is a running machine experiment result, C is a total arm feeding frequency result, and D is a rotation rate result.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Human embryonic stem cell H9 cell line (H9 hESCs): wicell company product, cat number: WA09 (H9) -DL-7.
WS hMSC in the following examples were differentiated from WRN-targeted human embryonic stem cell H9 cell line, and the preparation method of the cells was as follows:
the gene fragments of the 15 th exon (2509-2617 th position of GenBank: NM_000553.4, updated on PRI 15-MAR-2015) and the 16 th exon (2618-2686 th position of GenBank: NM_000553.4, updated on PRI 15-MAR-2015) of WRN in human genome are obtained by utilizing a molecular cloning method, wherein the gene fragments comprise designed mutation sites and Neo resistance genes for positive cloning screening, and then the gene fragments are subjected to homologous recombination with a helper adenovirus vector PCIHDADGT-4 vector (recorded in a document "An HSV amplicon-based helper system for helper-dependent adenoviral vectoers.Shuji Kubo,et al.BBRC.2003.307(4):826-830). Infection of hESCs is realized by introducing the mutated WRN gene fragments into H9 hESCs by utilizing the principle of homologous recombination and replacing wild type gene fragments, so that in-situ inactivation of the WRN gene is realized, and then a human embryonic stem cell H9 cell line of targeted knockdown WRN is finally obtained by utilizing the principle of homologous recombination, namely, the mutated N gene fragments are introduced into H9 hESCs and replaced by utilizing the principle of homologous recombination, so that in-situ inactivation of the WRN gene fragments is realized in the wild type WRN gene fragment is recorded in a document -/-hESC.WRN-/-.
WRN -/- hESC was subjected to Embryoid Body (EB) differentiation for 48-72 hours to obtain Embryoid Body (EB). EB was then inoculated into Matrigel (Invitrogen company product) coated six well plates for culture, and culture was continued for two weeks until the appearance of fibrous cells. After a further passage, the cell populations with positive CD73, CD90 and CD105 were sorted by flow cytometry, i.e. human mesenchymal stem cells with lost WRN function (WRN -/-) (denoted WS hMSC). Meanwhile, the H9 cell line is directionally induced and differentiated into mesenchymal stem cells, namely WT hMSC.
The HGPS hMSC in the following examples was differentiated from the human embryonic stem cell H9 cell line of LMNA (p.G608G) mutant (exon 1824C > T), and the preparation method of the cells was as follows:
The DNA fragment of exon 11 of LMNA gene was obtained by PCR amplification using LMNA gene correction vector as a template, and then cloned into pCR2.1-TOPO vector (Invitrogen). Point mutation of p.G608G (1824C > T) was achieved by using Invitrogen Point mutation kit GeneTailorSite-Directed Mutagenesis System, and the mutated DNA fragment was obtained by substituting the original wild type LMNA fragment in the LMNA gene correction vector. Thus, a recombinant plasmid having a mutation of LMNA (p.G608G) was obtained. Then, the recombinant plasmid carrying the LMNA (p.G608G) mutation was subjected to the PI-SceI enzyme tangential digestion, and then, the recombinant plasmid was introduced into a 116 cell line derived from a human embryonic kidney epithelial cell line (HEK 293) together with a helper virus AdHPBGF (described in document "Genome Size and Structure determine efficiency of postinernalization steps and gene transfer of capsid-modified adenovirus vectors in a cell-type-specific manner.Journal of Virology.2004.78(18):10009-10022")) to carry out packaging of adenovirus (AdV), the cell culture supernatant infected with AdV was collected, and the recombinant AdV carrying the LMNA (p.G608G) mutation was obtained by subjecting the recombinant plasmid to ultra-high-speed centrifugation, enrichment and purification, and then, the purified recombinant virus was subjected to the H9-hESC infection, and the hESC carrying the LMNA heterozygous mutation (G608G/+) was obtained by screening.
And (3) performing Embryoid Body (EB) differentiation on the hESC with the LMNA heterozygous mutation (G608G/+) for 48-72 hours to obtain Embryoid Body (EB). EB was then inoculated into Matrigel (Invitrogen company product) coated six well plates for culture, and culture was continued for two weeks until the appearance of fibrous cells. After a further passage, the cell populations with positive CD73, CD90 and CD105 were sorted by flow cytometry, namely human mesenchymal stem cells (designated HGPS hMSC) with the LMNA heterozygous mutation (G608G/+). While the H9 cell line was directionally induced to differentiate into mesenchymal stem cells (designated WT hMSC). HGPS hMSC is described in document "Wu Z,Zhang W,Song M,Wang W,Wei G,Li W,Lei J,Huang Y,Sang Y,Chan P et.al.Differential stemcell aging kinetics in Hutchinson–Gilford progeria syndrome and Werner syndrome.Protein Cell.2018.9(4):333-350".
The mesenchymal stem cell culture medium (MSC-CM) in the following examples was formulated as follows:
MEM Medium (Invitrogen corporation, cat# 12571071);
10% (volume percent) fetal bovine serum (Gibco company product);
0.1mM non-essential amino acid (Invitrogen company product, cat# 11140-050);
1mM GlutaMAX dipeptide (Invitrogen company product, cat# 35050-061);
1% (volume percent) (1 g/100 ml) penicillin/streptomycin (Invitrogen company product, cat# 15070-063);
10ng/ml human FGF2 (Joint Protein Central company).
The viral vectors expressing Luciferase in the examples described below are described in literature "Pan,H.,Guan,D.,Liu,X.,Li,J.,Wang,L.,Wu,J.,Zhou,J.,Zhang,W.,Ren,R.,Li,Y.,et al.(2016).SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2.Cell research 26,190-205.", and the biological material is publicly available from the applicant, and is used only for the experiments related to the repeated invention, and is not used for other purposes.
Example 1 screening of FDA drug library based on the Stem cell model for premature aging
In order to find more anti-aging drugs, 1622 compounds in the FDA drug library (selleck, L1300) were phenotypically screened, which included: WS hMSC were plated in 96-well plates at a density of 3,000 cells per well, and the hMSC medium containing 1. Mu.M drug was replaced on the second day, and the culture was performed once every other day for about six days. During this process, the growth of cells was recorded using the IncuCyte S3 living cell imaging system (Essen BioScience, MI USA) and the relative densities of cells were analyzed by imaging. The effect of each compound on WS hMSC cell proliferation was assessed by normalizing the cell relative density values of the drug-treated group (day 6) to the control-treated group (n=3).
The results show that gallic acid (GALLIC ACID, GA) can promote WS hMSC cell proliferation. The gallic acid has a structural formula shown in formula 1:
Example 2 detection of senescence phenotype after gallic acid treatment of WS hMSC
To examine the effect of gallic acid on WS hMSC senescence phenotype. The WS hMSC cells were cultured for 1 generation (7 days) with 1. Mu.M gallic acid intervention (i.e., the cultured WS hMSC system was added with gallic acid for 7 days, the concentration of gallic acid was 1. Mu.M), and the resulting cells were 1. Mu.M gallic acid-treated WS hMSC cells (designated GA-WS hMSC); the solvent 0.1% dimethyl sulfoxide (vehicle) is used for intervening and culturing WS hMSC cells for more than 1 generation (7 days) (namely, the dimethyl sulfoxide is added into a system for culturing WS hMSC for culturing, the volume percentage concentration of the dimethyl sulfoxide is 0.1%), and the obtained cells are the WS hMSC cells treated by the solvent (marked as Veh-WS hMSC). Changes in senescence-associated phenotypes were detected using GA-WS hMSC and Veh-WS hMSC (as controls) as test cells. The specific method comprises the following steps:
1. Cell growth curve assay
The growth capacities of the serially passaged Veh-WS hMSC cells and GA-WS hMSC cells were counted using cell counts, and the results showed that GA-WS hMSC exhibited a significant increase in proliferation capacity as compared to the Veh-WS hMSC cells (FIG. 1A). The specific operation steps are as follows:
1) Counting the cell accumulation proliferation times of the Veh-WS hMSC and GA-WS hMSC which are continuously passed through the cell counting;
2) Calculating the proliferation times of each generation of cells = the number of cells at the end of each generation/the number of cells at the start of each generation;
3) Cell cumulative proliferation fold = log 2 (fold P1 cell proliferation fold) +log 2 (fold P2 cell proliferation fold) + … +log 2 (fold P17 cell proliferation fold).
The statistical results are shown in fig. 1a, which shows that: GA-WS hMSC has enhanced proliferation capacity compared to Veh-WS hMSC cells, and is characterized in that when the proliferation rate of Veh-WS hMSC is slowed or even stopped, GA-WS hMSC can still maintain a faster proliferation rate for several generations, and the number of generations to achieve growth retardation is later than that of Veh-WS hMSC.
2. Monoclonal formation assay to detect cell proliferation
WS hMSC treated with gallic acid and solvent were stained with 10% crystal violet, and the results showed that GA-WS hMSC exhibited significantly increased proliferation potency compared to Veh-WS hMSC (FIG. 1B). The specific operation steps are as follows:
1) WS hMSC were seeded in 12-well plates at a density of 2000 cells/well, treated with gallic acid and solvent every other day.
2) After about 12 days of incubation, 4% PFA was fixed for 30 min. The PBS was washed twice.
3) 10% Crystal violet stain for 1hr.
4) Washing the excess dye liquor with clear water and airing.
5) The scanner scans, and the software image J calculates the gray value.
The statistical results are shown in fig. 1B, and the results indicate that: GA-WS hMSC has accelerated proliferation capacity compared with veh-WS hMSC, and is specifically expressed as follows: the GA-WS hMSC clone formed gray values greater than veh-WS hMSC.
3. SA-beta-Gal staining
Cell senescence-associated beta-galactosidase staining is a method for staining senescent cells or tissues based on up-regulation of SA-beta-Gal (senescence-associated beta-galactosidase) activity levels at the time of senescence. When X-Gal is used as a substrate, cells can generate a dark blue product under the catalysis of aging-specific beta-galactosidase. SA- β -Gal staining was performed on Veh-WS hMSC and GA-WS hMSC, respectively, as follows:
1) Seeding cells at appropriate density in six well plates;
2) When the cell density reaches 60-80%, collecting cells and washing twice with PBS;
3) 2% paraformaldehyde+0.2% isovaleraldehyde for 5 min;
4) PBS was washed twice;
5) Staining solution was added and incubated overnight at 37℃in the absence of light. Wherein the dyeing liquid comprises the following formula: 40mM citrate/sodium phosphate buffer (PH＝6)+100mM K4[Fe(CN)6]·3H2O+100mM K3[Fe(CN)6]+5M NaCl+1M MgCl+1mg/ml X-Gal+H2O.
6) PBS was washed twice;
7) Hoechst 33342 (Life technology company product, cat: h3570 Incubation for 5 minutes at room temperature in dark;
8) Washing with PBS once;
9) And observing and photographing under a microscope. And further quantitatively and statistically analyzing the ratio of SA-beta-Gal staining positive cells in the two groups of cells.
As a result, the ratio of SA-. Beta. -Gal staining positive cells was significantly reduced (P < 0.01) as compared with Veh-WS hMSC, as shown in FIG. 1C. It can be seen that the senescence process of GA-WS hMSC was significantly slowed down compared to Veh-WS hMSC.
4. Western blot detects the expression of P16, P21, LAP2 beta and LaminB1 proteins.
Firstly, extracting the total cell proteins of a control group and a treatment group, and detecting the protein expressed by the cells by using Western blotting. The primary antibody used was the P16 antibody (anti-P16, murine, BD Biosciences product, cat# 550834), the P21 antibody (anti-P21, rabbit, CELL SIGNALING Technology product, cat# 2947S), the LAP2 beta antibody (anti-LAP 2 beta, murine, CELL SIGNALING Technology product, cat# cat 611000, cat# laminB1, rabbit, abcam, ab 16048).
The secondary antibody was an HRP-labeled goat anti-rabbit antibody (Santa cruz, cat# sc-2005). GAPDH is used as an internal reference, a primary antibody is a murine anti-GAPDH antibody (Santa cruz company product, cat# sc-376248), and a secondary antibody is an HRP-labeled goat anti-mouse antibody (Santa cruz company product, cat# sc-2005).
The results of Western blotting detection of P16, P21, LAP2 beta and LaminB1 proteins are shown in FIG. 1D,
Compared with the control group, the gray level of P16 and P21 protein bands in WS hMSC treated by gallic acid is reduced, and the gray level of LAP2 beta and LaminB1 protein bands is increased. This suggests that gallic acid can down-regulate the levels of the senescence-associated markers P16, P21 proteins, up-regulate the levels of the nuclear membrane associated proteins LAP2 beta and LaminB1 proteins.
5. Cell cycle detection
The cell cycle is in turn divided into the intercellular and mitotic phases. The cellular intervals are further divided into G1, S and G2 phases according to the dynamic process of DNA synthesis. Propidium Iodide (PI) is a DNA dye that can be inserted into double-stranded DNA, and cells in the G1/S, S, G2/M phase can be identified and counted by detecting the fluorescence intensity by a flow analyzer. The specific experimental steps are as follows:
1) At 60% cell density, cells were digested with TrypLE.
2) Rinsing with PBS for 2 times, centrifuging at 1000rpm at room temperature for 5min, taking out supernatant, adding precooled 70% ethanol, and standing in a refrigerator at-20deg.C overnight.
3) The next day, PBS was added to the fixative, mixed upside down, and centrifuged at 3000rpm for 10min at room temperature.
4) The supernatant was discarded, PI staining solution was added, the cells were resuspended, and the mixture was placed in a 37℃water bath, and stained by incubation for 30min. Wherein PI staining solution formulation was analyzed by flow cytometry (BD, FACSCalibur) as follows ：0.1％TritonTM X-100(in PBS),0.02mg/mL Propidium iodide(Invitrogen,Cat.#P3566),0.2mg/mL RNase..
The results are shown in fig. 1E, where the proportion of S-phase cells is higher in the gallic acid treated group than in the control group, indicating that gallic acid can regulate the cell cycle of WS hMSC, reversing the aging-related changes in cell growth arrest.
6. Detection of telomere length
Telomeres are highly repetitive sequences of DNA located at the ends of chromosomes and are closely related to cell longevity, with higher aging levels leading to shorter telomere lengths. The detection is carried out according to the following steps:
(1) DNA extraction: the procedure was based on TIANGEN cell genomic DNA extraction kit instructions.
1) Adding 200 μl of GA buffer to the harvested cells, and uniformly resuspending the cells;
2) Adding 20 μl of protein kinase K and mixing thoroughly;
3) Adding 200 μl of GB buffer, mixing, and placing into 70 deg.C metal bath for cracking for 10min;
4) Adding 200 μl of absolute ethanol, mixing, covering the mixed solution in an adsorption column with 2ml of collecting tube, centrifuging at 12000rpm at room temperature for 1min, and discarding the waste liquid;
5) Adding 500 μl GD buffer solution into the adsorption column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
6) 600 μl of rinse BW was added, the column was washed twice, centrifuged at 12000rpm for 30s, the waste was discarded, and the column was centrifuged again for 2min.
7) The column was transferred to a fresh EP tube and 50-100. Mu.l of ddH2O was added to the very center of the built-in membrane of the column. After standing at room temperature for 5min, the mixture was centrifuged at 12000rpm for 2min, and the concentration of the obtained DNA was measured.
(2) QPCR detection
QPCR MASTER Mix (2×) kit, 384 well plates, 5 μl system:
1) Thawing 2× QPCR MASTER Mix on ice, mixing, and placing on ice all the time during use.
2) The extracted DNA was placed on ice, 0.2. Mu.l of the reverse transcription product and 1.9. Mu.l of ddH 2 O were mixed to prepare solution A, which was applied to the wells of the corresponding 384-well plate with a continuous applicator gun, respectively, 2.1. Mu.l/well, and repeated four times.
3) 2.5. Mu.l of 2X QPCR MASTER Mix and 0.2. Mu.l of each of the forward and reverse primers were mixed to give solution B, which was applied to the wells of the corresponding 384 well plates with a continuous applicator, respectively, in 2.9. Mu.l/well, and repeated.
4) Mixing A, B solutions, sealing, and centrifuging at low speed for 1min.
5) Quantitative detection was performed in real time using a qPCR instrument. Program setting:
95℃ 3min
95℃ 10s
44 cycles
55℃ 30s
65℃ 5s
Dissolution curve at 95℃for 5s 65-95℃for 5s
The qPCR primer sequences used were as follows:
Tel-F(5'-3')CAGAGACACACATAGGCTCAAA；
Tel-R(5'-3')AATCTGGGTGCTCCTGTATTG。
Internal parameters:
36B4u-F(5'-3')CAGCAAGTGGGAAGGTGTAATCC；
36B4d-R(5'--3')CCCATTCTATCATCAACGGGTACAA。
As a result, the content of telomere fragment in GA-WS HMSC DNA was increased as compared to Veh-WS hMSC, as shown in FIG. 1F. This suggests that gallic acid may slow telomere shortening.
Example 3 detection of the influence of gallic acid on WS hMSC pluripotent Stem function
The effect of gallic acid on WS hMSC multipotent stem function was examined using GA-WS hMSC and Veh-WS hMSC obtained in example 2 as control cells. The method comprises the following steps:
1. in vivo retention capacity assay of gallic acid-treated WS hMSC cell mice
To verify the effect of gallic acid on the persistence capacity of WS hMSC, veh-WS hMSC and GA-WS hMSC cells were first infected with a viral vector expressing Luciferase, respectively, and after 3-5 days of infection, the two cells were each digested into single cell state, and then injected into immunodeficient NOD-SCID mice (product of Peking Vitoliher laboratory animal technology Co., ltd.) on the left and right tibialis anterior muscle, respectively, at the same injection amounts. The in vivo retention capacity of hmscs was reflected by measuring the luciferase activity in the left and right tibialis anterior of mice, respectively, using a small animal in vivo imaging system (Xenogen IVIS spectrum, product of PE company) after 0-4 days of injection.
The specific operation method is as follows:
1) Selecting cells with good growth state and cell density of about 60-80%, and infecting a viral vector expressing Luciferase;
2) 3-5 days after infection, after the cells grow up, recording as day 0, and using TrypLExpress to digest into single cells;
3) Veh-WS hMSC and GA-WS hMSC cell counts, 5X 10 5 cells were resuspended in 50 μl PBS as one unit;
4) Equal amounts of cells were mixed with Luciferase substrate (D-Luciferin Firefly, product of the company GOLDBIO) and the relationship between fluorescence intensity and cell number was measured using an enzyme-labeled instrument.
5) Taking 50 μl of cell suspension, and respectively injecting into tibialis anterior of the mice;
6) The state of the mice is observed every day;
7) After 0,1, 2,3,4 days post-implantation, mice were removed and analyzed with a small animal in vivo imaging system after intraperitoneal injection of Luciferase substrate and gas anesthesia. Fluorescence intensities were counted using 5 groups of biological replicates.
The results showed that GA-WS hMSC (right leg) had significantly enhanced activity in tibialis anterior compared to Veh-WS hMSC (left leg) (A in FIG. 2), indicating that GA-WS hMSC has a slower rate of in vivo retrograde and increased retention compared to Veh-WS hMSC.
2. Detection of the Capacity of gallic acid-treated WS hMSCs to three-line differentiation
(1) Differentiation into adipocytes
To determine the potential of gallic acid treated WS hMSCs (i.e., GA-WS hMSC cells) to differentiate into adipocytes, and using Veh-WS hMSC as a control, the following procedure was followed:
1) Cells were seeded at a density of 3×10 5 on a 0.1% gelatin coated 12 well plate and nearly fused the next day to ensure that no density differences between cell lines were generated due to growth rate.
2) Differentiation was initiated by adding 1/1000 of 10mg/ml Polybrene (Sigma, 107689), and 4. Mu. L rttA virus with 1. Mu.L PPARG virus (rtta and PPARG virus are described in literature "T.Ahfeldt et al.,Programming human pluripotent stem cells into white and brown adipocytes.2012.Nat Cell Biol 14,209") to infect cells. 1/1000 of 2mg/ml Doxycycline (Clontech, 1486c 511) was added simultaneously for induction.
3) After 24h of virus infection, MSC medium was changed and the induction was continued by adding Dox.
4) After 48h of virus infection, the fat medium was changed for cultivation and fresh medium was changed once for 3-4 days, and the Dox was continued twice. About 30 days, the formation of lipid droplets in the form of particles was observed.
5) At this time, the lipid droplets easily float, the medium is gently discarded, and the medium is washed with PBS for 2 times. After fixing 4% paraformaldehyde at room temperature for 20min, staining with oil red-O solution for 30min, and washing with PBS for 2 times. Observation under an inverted microscope and photographing. After that, the absorbance was measured at 510nm after dissolution in isopropanol.
The culture medium is a fat culture medium, and the formula of the fat culture medium is as follows: 84% high sugar DMEM (HyClone, SH 30243.01), 7.5% Knock-Out Serum Replacement (Thermo, A3181502), 7.5% human serum albumin solution (Solarbio,A8230),0.5％MEM NEAA(Gibco,11140076),0.5％Peniciin-Streptomycin(Gibco,15140-163),0.1μM Dexamethasone(Selleck,S1322),10μg/mL Insulin(Sigma,91077C),0.5μM Rosiglitazone(Selleck,S2556),1/10000Plasmocin(InvivoGen,ant-mpp).
As the results in FIG. 2B show, GA-hMSC increased absorbance values compared to Veh-hMSC, and more GA-hMSC lipid droplets were observed visually than Veh-hMSC. This suggests that intervention with gallic acid may activate the potential of WS hMSC to differentiate into adipocytes compared to control treatments.
(2) Differentiation into osteoblasts
To determine the potential of gallic acid-treated WS hMSCs (i.e., GA-WS hMSC cells) to differentiate into osteoblasts, and using Veh-WS hMSC as a control, the following procedure was followed:
1) GA-WS hMSC and Veh-WS hMSC were plated at 1X 10 5 on 0.1% gelatin coated 6-well plates, respectively, and cultured with MSC-CM until cells were completely fused.
2) The cells are cultured by an osteogenic differentiation medium, the fresh medium is gently replaced for 3 to 5 days, and gray calcium deposition on the surfaces of the cells can be observed after about 30 days.
3) Staining was performed with Von kossa staining kit.
4) Irradiation with a UV cross-linker, blackening of calcium deposit, observation with an inverted microscope and photographing. Grey values were determined using ImageJ.
Osteogenic differentiation medium formula ：88％MEMα(Gibco,32571-101),1％GlutaMAX(Gibco,35050079),1％Peniciin-Streptomycin(Gibco,15140-163),10％FBS(Gibco,42F1190K),10mMβ-glycerolphosphate(Sigma,G9422),0.1μM Dexametasome(Selleck,S1322),50μg/mL Ascorbic Acid(Sigma,A4403),1/10000Plasmocin(InvivoGen,ant-mpp).
As shown by the results in FIG. 2B, the GA-hMSC greyscale value was increased over Veh-hMSC, and the GA-hMSC calcium deposition area was observed to be larger than Veh-hMSC under the mirror. This suggests that intervention with gallic acid may activate the potential of WS hMSC to differentiate into osteoblasts compared to control treatments.
(3) Differentiation into chondrocytes
To determine the potential of gallic acid-treated WS hMSCs (i.e., GA-WS hMSC cells) to differentiate into chondrocytes, the following procedure was followed:
1) WS hMSC at a density of 1X 10 5 was inoculated into a low adsorption 96-well plate, and 100. Mu.L of cartilage medium was added to each well.
2) The plates were placed in a centrifuge and centrifuged at 450g for 5min at room temperature, after which cell clumping was observed.
3) Cartilage medium was changed every 2-3 days and cultured for about 30 days.
4) The size of the cartilage ball was recorded by photographing under a microscope.
5) After observing the sphere diameter, fixing with 4% paraformaldehyde and dehydrating with 30% sucrose solution, embedding with OCT.
6) The slices were serially sectioned with a frozen microtome to a thickness of 0.12. Mu.m.
7) The complete maximum cross-sectional slice was selected and placed on a polylysine-treated slide.
8) The embedding agent is removed by washing twice with PBS and is dyed with toluidine blue dye liquor for 30min. Excess staining solution was washed off with PBS and the staining effect was observed under an inverted microscope.
The formula of the cartilage culture medium comprises: 96% high sugar DMEM(HyClone,SH30243.01),1％Glutamax(Gibco,35050079),1％MEMNEAA(Gibco,11140076),1％Peniciin-Streptomycin(Gibco,15140-163),100nM Dexamethasone(Selleck,S1322),1％ITS,50μg/mL Ascorbic acid(Sigma,A4403),40μg/mL Proline(Sigma,P5607).
As shown by the results in FIG. 2B, GA-hMSC formed cartilage balls with significantly increased diameters compared to Veh-hMSC, and GA-hMSC cartilage balls were observed visually to be larger than Veh-hMSC. This suggests that intervention with gallic acid may activate the potential of WS hMSC to differentiate into chondrocytes compared to control treatments.
Example 4 modulation of WS hMSC mitochondrial homeostasis by gallic acid.
The WS hMSC cells are cultivated by 1 mu M gallic acid for 2 generations (14 days) (namely, gallic acid is added into a system for cultivating the WS hMSC for cultivation, the concentration of the gallic acid is 1 mu M), and the obtained cells are WS hMSC cells treated by 1 mu M gallic acid (marked as GA-WS hMSC); the WS hMSC cells were cultured by solvent 0.1% dimethyl sulfoxide (vehicle) for 2 passages (14 days) (i.e., the culture was performed by adding dimethyl sulfoxide to the system for culturing WS hMSC, the volume percentage concentration of dimethyl sulfoxide was 0.1%), and the cells were solvent-treated WS hMSC cells (designated as Veh-WS hMSC). The resulting GA-WS hMSC and Veh-WS hMSC (as controls) were used as test cells to examine the regulation of WS hMSC mitochondrial homeostasis by gallic acid. The method comprises the following steps:
1. Detection of mitochondrial reactive oxygen species (Reactive oxygen stress, ROS)
As cells age, the level of reactive oxygen species in the mitochondria increases. Mitochondrial ROS levels were flow analyzed after staining with Mitosox Red (Invitrogen, cat. # M36008). The specific method comprises the following steps:
1) After the test cells were digested with TrypLE, they were collected into a 1.5mL EP tube.
2) Cells were resuspended in 300. Mu.L of 2.5. Mu.M Mitosox Red staining solution.
3) And (5) dyeing for 30min at room temperature in dark.
4) And the mixture is centrifuged at 1000rpm for 5min at room temperature. The supernatant was discarded and the cells were resuspended in 500. Mu.L PBS.
5) Mitochondrial ROS levels of hMSCs were detected by flow cytometry (BD LSRFortessa).
As the results in fig. 3 a show, the average fluorescence intensity of the gallic acid-treated group was lower than that of the control group, indicating that gallic acid can reduce the level of ROS in mitochondria.
2. Detection of mitochondrial mass
Mitochondrial mass increased with the onset of the aging process, and mitochondrial mass levels were analyzed using a flow cytometer after staining with acridine orange 10-bromononane (Nonyl Acridine Orange (NAO), invitrogen, cat.#a1372).
1) After the test cells were digested with TrypLE, they were collected into a 1.5mL EP tube.
2) Cells were resuspended in 500. Mu.L of 10. Mu.M NAO staining solution.
3) Light was protected from the light and stained in a cell incubator with 5% CO2 at 37℃for 10min.
4) And the mixture is centrifuged at 1000rpm for 5min at room temperature. The supernatant was discarded and the cells were resuspended in 500. Mu.L PBS.
5) Mitochondrial mass of hMSCs was detected with a flow cytometer (BD LSRFortessa).
As the B results in fig. 3 show, the average fluorescence intensity of the gallic acid-treated group was lower than that of the control group, indicating that gallic acid can reduce the level of mitochondrial quality.
3. Detection of mitochondrial membrane potential
During oxidation respiration, mitochondria store the generated energy in the form of electrochemical potential energy in the inner mitochondrial membrane, and the transmembrane potential generated by the asymmetric distribution of hydrogen ions and other ions formed on both sides of the inner mitochondrial membrane is called mitochondrial membrane potential. During cell senescence, the membrane potential decreases. Using a commercial JC-10 kit: CELL METER TM JC-10Mitochondrial Membrane Potential Assay Kit (AAT Bioquest, cat. # 22801) detected the mitochondrial membrane potential of hMSC. The specific experimental steps are as follows:
1) After the test cells were digested with TrypLE, they were collected in a 1.5mL EP tube.
2) And mixing the solution A and the solution B in the JC-10 detection kit according to the proportion of 1:200 to prepare JC-10 working solution.
3) The cells were resuspended in 500. Mu.L JC-10 working solution, protected from light, and stained in a cell incubator with 5% CO 2 at 37℃for 30min.
4) Mitochondrial membrane potential of hMSCs was detected with a flow cytometer (BD LSRFortessa). Mitochondrial membrane potential is indicated by the intensity of green and red fluorescence in the cell, mitochondrial membrane potential=fl (590 nm)/FL (530 nm).
As shown in fig. 3C, FL (590 nm)/FL (530 nm) was higher in the gallic acid-treated group than in the control group, suggesting that gallic acid may up-regulate mitochondrial membrane potential.
Example 5, results of experiments on gallic acid-alleviating multiple aging cell models.
The senescent cell model (i.e., the test cell) selected in this example was: hutchinson-Gilford progeria syndrome (HGPS) hMSC differentiated from the LMNA gene mutant H9 hESC (noted HGPS hMSC), WT hMSC (directly differentiated from H9 hESC) (noted RS WT hMSC) that resulted in replicative senescence (REPLICATIVE SENESCENCE, RS) were continuously passaged, aged WT hMSC induced at an Ultraviolet (UV) intensity of 300 μJ/cm2, aged WT hMSC induced by 50 μM hydrogen peroxide (H 2O2), and human aortic endothelial cells (human arterial endothelial cell, hAEC) were continuously passaged to result in replicative aged hAEC (noted RS hAEC).
The preparation method of the RS WT hMSC comprises the following steps: after 12 passages of serial subculture of WT hMSC, replicative aged WT hMSC was obtained.
The preparation of UV or H 2O2 induced WT hMSC cells was as follows:
The preparation of UV-induced senescent WT hMSC cells was as follows: WT hMSC was plated in six well plates at 30,000/well, and after two days of culture, the cell density was about 60%, the medium was aspirated, and the medium was replaced with a new medium after irradiation with Ultraviolet (UV) light at 300. Mu.J/cm 2. Thus, UV-induced aged WT hMSC was obtained.
The preparation of H 2O2 -induced senescent WT hMSC cells was as follows: WT hMSC was plated in six-well plates at 30,000/well, and after two days of culture, the cell density was about 60%, and after treatment of cells with 50. Mu.M hydrogen peroxide for 24 hours, the normal medium was changed. Thus obtaining the H 2O2 -induced aging WT hMSC cells.
The preparation method of the RS hAEC comprises the following steps: after continuous culture of the primary isolated human aortic endothelial cells for 8 passages, replicative aged hAEC was obtained.
1. Monoclonal formation assay to detect cell proliferation
Cells to be tested were seeded into 12-well plates at a density of 2000/well and treated with gallic acid or solvent every other day. The steps of gallic acid treatment of cells were as follows: 1 mu M gallic acid is used for intervening and culturing the cells to be tested for 1 generation (7 days) (namely, the gallic acid is added into a system for culturing the cells to be tested for 7 days, and the concentration of the gallic acid is 1 mu M), so as to obtain cells treated by the gallic acid; and (3) intervening in culturing the cells to be detected for 1 generation (7 days) by using a solvent of 0.1% dimethyl sulfoxide (vehicle) (namely adding dimethyl sulfoxide into a system for culturing the cells to be detected for culturing, wherein the volume percentage concentration of the dimethyl sulfoxide is 0.1%), and obtaining the cells which are treated by the vehicle.
Each of the obtained gallic acid-treated cells was subjected to a monoclonal formation assay as described in "example 2 step 2" above, and each of the vehicle-treated cells was examined for its ability to proliferate.
The results are shown in fig. 4a, which shows that: gallic acid can increase proliferation capacity of HGPS hMSC, RS WT hMSC, and UV or H 2O2 induced WT hMSC. The concrete steps are as follows: after gallic acid treatment, the grey scale values formed by HGPS hMSC, RS WT hMSC, and UV or H 2O2 induced WT hMSC clones were greater than the control.
2. SA-beta-Gal staining
The gallic acid-treated cells obtained in step 1 and vehicle-treated cells were stained for SA-. Beta. -Gal, respectively, according to the SA-. Beta. -Gal staining described in "example 2, step 3" above.
The results are shown in fig. 4B and C, with significantly reduced SA- β -Gal staining positive cell ratios and significant differences in statistical analysis compared to the control group. It follows that gallic acid can delay the senescence phenotype of HGPS hMSC, RS WT hMSC, UV or H 2O2 induced WT hMS and hAEC.
Example 6 transcriptome analysis of gallic acid regulation of WS hMSC and HGPS hMSC gene expression.
Culturing WS hMSC with culture medium containing 1 μm gallic acid for three generations, culturing HGPS hMSC for two generations, collecting cells, extracting cell RNA, and extracting RNA by the following method:
1) Cells were digested and collected, centrifuged at 1000 rpm for 5 minutes;
2) 1ml Trizol was added to the cells, and the mixture was stirred and left at room temperature for 5min.
3) 200. Mu.l of chloroform was added, and after thoroughly mixing the mixture upside down, the mixture was left at room temperature for 15min, and was centrifuged at 12000g at 4℃for 15min.
4) The upper aqueous phase was pipetted into another centrifuge tube (RNA in upper aqueous phase, DNA in middle phase, protein in phenol phase).
5) 500. Mu.l of isopropanol was added, mixed well and left at room temperature for 10min, centrifuge at 4℃for 12000g,10min.
6) The supernatant was discarded, RNA was immersed in the bottom of the tube, 1ml of pre-chilled 75% alcohol was added, the pellet was suspended by gentle shaking, and centrifuged at 12000g for 5min at 4 ℃.
7) The supernatant was discarded as much as possible, and the mixture was dried at room temperature for 5min, and the RNA sample was dissolved in 20. Mu.l of RNase-free water at room temperature for 5min.
8) RNA concentration was measured using a Synergy H1 full-function microplate detector. The RNA was then sent to the testing company for RNA seq transcriptome detection.
As shown in figure 5a, gallic acid treatment resulted in altered levels of the transcriptome of WS hMSC and HGPS hMSC, and enrichment of the differential gene found that gallic acid up-regulated cell proliferation-related items and down-regulated apoptosis-related items. Also shown in FIG. 5B, the expression of senescence-associated genes in both WS hMSC and HGPS hMSC was regulated by gallic acid. This suggests that a series of changes in gene expression mediate the effects of gallic acid in delaying human stem cell senescence.
Example 7 gallic acid can delay the development of a normal mouse senescence-associated phenotype.
All animal experiments were performed according to the protocol approved by the institutional animal care and use committee of chinese academy of sciences. Wild type C57BL/6J mice (18 months old, style Bei Fu (Beijing) Biotechnology Co., ltd.) were free to obtain normal food and water in a pathogen-free environment at 23-24℃under 12 hours of light and 12 hours of darkness. The cage padding is replaced once a week.
The C57BL/6J mice were randomly divided into two groups, namely, a GA group and a control group, each group of 20 animals, and each group was continuously dosed for 12 months as follows.
GA group: the gallic acid is dissolved in water and is administrated in a drinking water mode every day, and the administration dosage is 100 mg/kg/day;
Control group: normal drinking water is administered.
During the administration period, the mice were subjected to phenotypic measurement such as grip force and different experiments such as running machine and Y maze. All data were counted using independent sample t-test processing with SPSS12.0 (SPSS inc., USA) statistical software. The experimental methods are as follows:
(1) Grip measurement
The right hand holds the mouse and presses the back and put on the holding power board of holding power appearance, and the left hand pushes forward and holds the holding power board, then the right hand backward slides to the portion of the rat tail, and the left hand is light to hold the holding power board, and the holding power board slides forward along with the strength of right hand pull the rat tail, and the afterburning of afterburning is acted on when the mouse is held the holding power board with effort, detects the biggest holding power of this mouse. Each mouse measures 10 grabbing force values each time, and the average value of the 10 measurements is compared with the weight of the mouse to obtain a relative pulling force value for statistical analysis.
As shown in fig. 6a, the relative grip detected in the gallic acid-continuously treated mice at 3 months and 4 months of administration was significantly better than that of the control group mice, indicating that gallic acid can improve the maximum muscle strength in the aged mice.
(2) Running machine experiment
The administration was continued for 10 months. Mice were trained on the treadmill for 3 consecutive days. The initial speed is 5m/min, and after 5min, the speed is continuously accelerated to the exhaustion of the mice (the electrodes are continuously shocked for more than 5 s) at 1m/min 2. And then performing a formal experiment, setting the electrical stimulation to be 2mA, recording the total movement distance detected when the mice are exhausted, and performing statistical analysis.
As shown in fig. 6B, the total distance moved by the mice in the gallic acid-treated group was greater overall than that of the mice in the control group, indicating that gallic acid can increase the motor ability of the aged mice.
(3) Y maze
The administration was continued for 11 months. The mice were scored for arm entry (number and order) over 5min, counted for total arm entry and number of rotations (three different arms were entered one time in succession) and each statistically analyzed.
As shown in fig. 6C and fig. 6D, the total arm feeding times and the rotation rate of the mice continuously treated with gallic acid were both superior to those of the mice in the control group, indicating that gallic acid has the effect of improving the memory of the aged mice.
In conclusion, gallic acid can delay the occurrence of the aging-related phenotype of normal mice.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (1)
1. The use of gallic acid as the sole active ingredient in the manufacture of a medicament for the treatment and/or prophylaxis of human premature ageing.
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