CN110330542B

CN110330542B - Compound for delaying human mesenchymal stem cell aging and preparation method and application thereof

Info

Publication number: CN110330542B
Application number: CN201910681834.6A
Authority: CN
Inventors: 肖建辉; 许艳
Original assignee: Affiliated Hospital of Zunyi Medical University
Current assignee: Affiliated Hospital of Zunyi Medical University
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2021-09-14
Anticipated expiration: 2039-08-19
Also published as: CN110330542A

Abstract

The invention relates to a compound for delaying human mesenchymal stem cell aging, a preparation method and an application thereof, wherein the compound is a triterpenoid compound ganoderic acid D, and the preparation method comprises the steps of drying ganoderma lucidum sporocarp, crushing the dried ganoderma lucidum sporocarp into fine powder, adding a proper amount of methanol for extraction to obtain an extract concentrated solution, drying the concentrated solution to obtain an extract, performing silica gel column chromatography, performing gradient elution by using chloroform-methanol, collecting eluent with a mobile phase of 70:30 gradient, performing silica gel column and ODS open column separation on the obtained eluent, and finally performing reverse phase semi-preparative HPLC separation to obtain the compound ganoderic acid D with a chemical molecular formula of C₃₀H₄₂O₇. The compound is extracted from lucid ganoderma sporocarp, has the activity of inhibiting stem cell senescence, has good application value for delaying stem cell senescence, and can be developed into a new anti-senescence medicine.

Description

Compound for delaying human mesenchymal stem cell aging and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a compound for delaying human mesenchymal stem cell senescence and a preparation method and application thereof.

Background

Mesenchymal Stem Cells (MSCs) are tissue Stem Cells with multipotentiality, and can proliferate and differentiate in the direction of osteogenic, cartilage, adipose, neural, and myocardial Cells. Based on the extensive plasticity of the cells, the cells are widely applied and researched in a plurality of animal disease models and clinical tests.

Aging is a natural law in the life development process, but from ancient times to present, people pursue and explore the mysterious secret of permanently keeping youth and prolonging life with cumin. Delaying aging and preventing aging-related diseases have become important issues facing human beings. Although "growing and aging" and prolonging the natural life (life span) are difficult to realize, it is feasible to delay aging, suppress the occurrence and development of aging-related diseases, prolong the health span (health span), realize healthy aging, and reduce the burden of aging-related diseases on the society. Cells are the basic unit of activity of a living body, and cellular senescence is the basis of tissue, organ and body senescence. Among the theories of aging of the body, the theory of stem cell aging is widely accepted in the industry. Stem cells are a type of cells having self-renewal and multipotential differentiation potential, exist in various tissues and organs of the body, and are an important source of self-renewal of aging or damaged cells in the tissues and organs. Thus, the senescence of an organism is in fact its Stem Cell senescence, and all senescence phenomena including tissue organ degeneration, loss of function, tumorigenesis and recurrent infections among the senile diseases reflect the level of Stem Cell senescence in the organism (Sharpless NE, Depinho RA. How Stem cells and low this makes us grow. Nat Rev Mol Cell Biol, 2007, 8(9): 703-713; Rosengardnten Y, Mckenna T, Grochov a D, et al. Stem Cell depletion in Hutchon-Gilford promoter syndrome. Aging Cell, 2011, 10(6): 1011-1020). The aging of stem cells causes the self-renewal and the multipotential capacity of the stem cells to be reduced, thereby causing the damaged tissues and organs to be difficult to repair and regenerate, and causing the reduction of the structures and functions of the tissues and organs and the development of diseases related to the aging. In conclusion, stem cells are the best model for studying Cell senescence, and the search for methods for delaying stem Cell senescence, reactivating stem cells and regulating their directed differentiation have important scientific significance and application value in the discovery of senescence-delaying agents, the prevention of geriatric diseases and the treatment of degenerative diseases (Ren R, Ocompo A, Liu GH, et al.

It has been found that Aging of Stem cells can lead to decreased wound healing capacity and loss of skin elasticity in mice (Rosengardnten Y, Mckenna T, Grochov. D, et al. Stem Cell depletion in Hutchinson-Gilford promoter syndrome. Aging Cell, 2011, 10(6): 1011) 1020), while rejuvenating Stem cells can reverse the body-related Aging phenotype (Rando TA, Chang HY. Aging, rejuvenation, and epigenetic reprogramming the Aging Cell 2012, 148: 46-57.). Thus, the aging depletion of stem cells in vivo may be the root cause of aging in the body (Ren R, Ocampo A, Liu GH, et al. Regulation of stem Cell formation by metabolism and epidemics. Cell Metab, 2017, 26(3): 460-474.). Mesenchymal stem cells are a class of adult stem cells with mesodermal lineage differentiation ability, which can be differentiated into osteogenic, cartilaginous and adipogenic cells and play an important role in tissue repair (Alviano F, Fossati V, Marchionni C, et al. Term, intracellular membrane is a high throughput source for multi-potential therapeutic stem cells with the ability to differentiate into endogenous cells in vitro. Bmc development Biology, 2007, 7(1): 11-25). Studies have shown that the decrease in the number and proliferation capacity of mesenchymal Stem cells occurs with the increase of the randomized somatic age of mesenchymal Stem cells (Liang R, Ghaffari S. Stem cells, Redox signaling, and Stem cell imaging. Artificial Redox Signal, 2014, 20: 1902. 1916. Stenderup K, Justesen J, Clausen C, et al. Aging associated with the deleted large Bone life span and accessed sensory of Bone marrow stromal cells, Bone marrow cells, 2003, 33: 919-), and accelerated depletion of mesenchymal Stem cells can be observed in both early senescence and senescent model mice, including Werner syndrome and transmitting Gilford syndrome (liver KR, Barkho, RuZ, Hu. 221. growth of liver cells, and Kidney growth of liver cells, kidney growth promoter 472. growth of liver cells, kang KS. Aging-related genes in mesenchyme stem cells a mini-review, Gerontology, 2013, 59: 557-. Furthermore, transplantation of mesoderm-derived Stem Cells into aging mice can extend their lifespan (Singh L, Brennan TA, Kim JH, et al, Brief report: long-term functional expression of sensory genetic promoter Cells in a mouse model of open-ended formation Stem Cells, 2013, 31:607 Cells 611). This means that the aging of the mesenchymal stem cells is closely related to the aging of the body, and the delaying of the aging of the mesenchymal stem cells is an important way for preventing and delaying the aging of the body.

Since natural products are an important source for creating new drugs, it has been found that some natural compounds such as polysaccharides, polyphenols, flavonoids, etc. have the function of delaying aging. For example, Monorden and Tanessporins have been reported to improve the health and prolong the life of the model organisms, caenorhabditis elegans (Georges E. Janssens, Xin-Xuan Lin, Llu i s Millan-Ari ñ o, et al. Transcriptomics-based screening identification of Hsp90 a means to selection. Cell Reports, 2019; 27: 467-.

Therefore, the inventor establishes a human mesenchymal stem cell aging model by taking natural compounds from rare Chinese medicinal materials such as ganoderma lucidum, cordyceps sinensis and the like as screening objects. Through a large amount of screening, the triterpene compound Ganoderic acid D (GA-D) is firstly discovered internationally, can obviously delay the aging of human mesenchymal stem cells, and has huge clinical application potential.

Disclosure of Invention

Aiming at the problems in the background art, the invention aims to provide a compound with the function of safely and efficiently delaying the aging of human mesenchymal stem cells based on the important scientific significance and clinical value of delaying the aging of stem cells in preventing aging-related diseases, in particular to a compound for delaying the aging of human mesenchymal stem cells and a preparation method and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a compound for delaying human mesenchymal stem cell aging, wherein the molecular formula of the compound is C₃₀H₄₂O₇The structural formula is shown as the general formula (I):

the invention relates to a preparation method of a compound for delaying human mesenchymal stem cell senescence, which comprises the following steps:

drying Ganoderma fruiting body, pulverizing into 200-300 mesh fine powder, extracting with 80-95% methanol for 3 times, collecting supernatant, concentrating under reduced pressure to obtain concentrated extract, drying the concentrated extract to obtain extract, subjecting the extract to silica gel column chromatography, gradient eluting with chloroform-methanol, collecting eluate with mobile phase of 70:30 gradient, and separating the eluate with silica gel column and ODS open columnFinally, separating by reverse phase semi-preparative HPLC to obtain the compound with the chemical molecular formula C₃₀H₄₂O₇The chemical structural formula of the compound is shown as the general formula (I).

The compound for delaying the aging of the human mesenchymal stem cells is applied to the preparation of the medicine for delaying the aging of the stem cells, the using dose concentration of the compound in the medicine is 0.001-100 mu M (micromolar concentration), the compound has no cytotoxicity on the human mesenchymal stem cells, the prepared medicine can reduce the number of beta-galactosidase positive cells, reduce the active oxygen content in the cells, reverse the growth cycle retardation of the cells, and enhance the activity of the telomerase of the cells.

The compound for delaying human mesenchymal stem cell senescence can be prepared into a medicine of a tablet, a capsule, a pill, a fat emulsion or an ointment together with medically acceptable pharmaceutic adjuvants, and the using dose concentration of the compound in the prepared medicine is 1-10 mu M (micromolar concentration).

The invention has the beneficial effects that: the compound extracted from the lucid ganoderma sporocarp has the inhibitory activity of inducing cell senescence, can reduce the number of beta-galactosidase positive cells, reduce the content of active oxygen in the cells, reverse cell growth cycle retardation and enhance the activity of cell telomerase, has good application value when being applied to delaying stem cell senescence, and can be developed into a new anti-senescence medicine.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings.

FIG. 1 is a comparison of the identification of hAMSCs: wherein A represents a comparison graph of morphological observation of P3 generation hAMSCs: (a) 40X, (b) 100X; b represents a characteristic protein comparison graph of the hAMSCs identified by immunocytochemistry staining: (a, b) a PBS control group, (c, d) an epithelial keratin group, (e, f) a vimentin group; scale bar: 50 μm, 20 μm; c represents a contrast chart of the molecular marker on the surface of the hAMSCs identified by flow cytometry;

FIG. 2 is an HPLC purity analysis chart of GA-D;

FIG. 3 is a graph of high resolution mass spectrometry analysis of GA-D;

FIG. 4 is a GA-D NMR hydrogen spectrum;

FIG. 5 is a GA-D NMR carbon spectrum;

FIG. 6 is a graph comparing the generation of GA-D inhibiting human mesenchymal stem cell senescence marker β -galactosidase with active oxygen: wherein A represents a staining pattern for beta-galactosidase: (a) a normal group, (b) a model group, (c) a 0.001. mu.M GA-D treatment group, (D) a 0.01. mu.M GA-D treatment group, (e) a 0.1. mu.M GA-D treatment group, (f) a 1. mu.M GA-D treatment group, (g) a 10. mu.M GA-D treatment group, (h) a 100. mu.M GA-D treatment group; b is a beta-galactosidase staining and positive cell proportion statistical analysis chart, and C is a comparison chart for detecting ROS content by a DCFH-DA method: (a) normal group, (b) model group, (c) 10 μ M GA-D treatment group, Scale bar: 100 μm;

FIG. 7 is a graph comparing GA-D inhibition of human mesenchymal stem cell growth cycle protein p21 expression and increased telomerase content: wherein A represents the expression and statistical analysis chart of p21 protein; b represents a telomerase content detection comparison graph;

FIG. 8 is a graph showing the effect of GA-D on cell viability.

Detailed Description

In order to fully explain the implementation of the present invention and to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of implementations of the invention and do not limit the invention.

Example 1: isolated culture and identification of human mesenchymal stem cells

Fresh placenta obtained by full-term caesarean section is placed in a sterile square dish, and blood stain on the surface of the placenta is cleaned by using sterile D-PBS (containing 1% double antibiotics), and the amnion is peeled off and cut into pieces. Transferring to a 50mL sterile centrifuge tube, adding pancreatin digestive juice (0.5% trypsin-0.2% EDTA-2 Na), digesting at 37 deg.C under rotation at 200rpm for 90min, and filtering with 300 mesh filter screen. Washing the digested amnion with D-PBS, transferring into another sterile centrifuge tube, adding collagenase digestive juice (0.5 mg/mLII type collagenase-0.05 mg/mL DNaseI) with the same volume as amnion, and performing rotary digestion at 37 deg.C and 200rpm for 50min until amnion is floccule. Filtering with 300 mesh filter screen to obtain filtrateCentrifuging at 1500rpm for 10min, discarding supernatant, adding LG-DMEM/F12 into the precipitate, and completely suspending to obtain primary human mesenchymal stem cells. The cells were seeded in T25 flasks. Washing with D-PBS for 3 times after two days, removing impurities, replacing with fresh culture medium, 37 deg.C, and 5% CO₂Culturing under saturation humidity, when the cell fusion degree reaches 80% or more, digesting for about 2min by using 0.125% trypsin-0.02% EDTA-2Na digestive juice until the cell morphology becomes round, tapping the bottle body to make the cell fall off, adding an equal volume of complete culture medium to stop digestion, centrifuging at 1000rpm for 5min, discarding the supernatant, adding a proper amount of LG-DMEM/F12 to completely culture the basic suspension cell and seeding the basic suspension cell, namely the 1 st generation human mesenchymal stem cell. Subsequent subculture, and the 2 nd to 5 th substitutes for subsequent experiments, the human mesenchymal stem cell grows in spindle shape, fiber shape and vortex shape, the specific situation is shown in figure 1A, the detection result of typical cell phenotype characteristics is shown in figure 1, the immunocytochemical detection shows that the human mesenchymal stem cell highly expresses a mesenchymal cell surface marker Vimentin (brown), does not express an epithelial cell marker keratin CK19, and the specific situation is shown in B in figure 1; flow cytometry detection results showed that it highly expressed mesenchymal cell surface molecules CD105 (88.10%), CD73 (99.95%), CD90 (98.48%), CD44 (99.89%) and CD29 (99.96%), did not express hematopoietic stem cell markers CD34, CD11b, CD19, CD45 and MHC-II molecules HLA-DR, as shown in fig. 1C.

Example 2: separation, preparation and identification of GA-D natural compound

Drying Ganoderma fruiting body, pulverizing into 200-mesh fine powder of 300 meshes, extracting with 80-95% methanol for 3 times, collecting supernatant, concentrating under reduced pressure to obtain extract concentrate, drying the concentrate to obtain extract, subjecting the extract to silica gel column chromatography, eluting with chloroform-methanol gradient, collecting eluate with 70:30 gradient mobile phase, separating the eluate with silica gel column and ODS open column, and separating with reverse phase semi-preparative HPLC to obtain ganoderic acid D with a structure formula (I) as white crystal with HPLC purity of 98% (as shown in FIG. 2), and analyzing with mass spectrometry to obtain a product with a purity of above 98% (as shown in FIG. 3)，ESI-MS:m/z 513.3[M-H]^-1. In NMR hydrogen spectroscopy (as shown in fig. 4), the main results of the high field region are δ 1.03 (3H, s), δ 1.25 (3H, s), δ 01.00 (3H, d, J =6.0 Hz), δ 11.19 (3H, d, J =5.1 Hz), δ 21.13 (3H, s), δ 31.10 (3H, s) and δ 41.32 (3H, s) are methyl hydrogen signals at

positions

18, 19, 21, 27, 28, 29 and 30, respectively, and the compound can be judged to be a tetracyclic triterpene; low field region δ 54.87 (1H, m) is the hydrogen signal on the hydroxyl carbon; in NMR carbon spectroscopy analysis (as shown in fig. 5), the low field regions δ 217.6, δ 216.6, δ 207.8, δ 197.6 and δ 180.9 are carbonyl carbon signals of 3, 15, 23, 11 and 26, respectively; δ 157.9 and δ 141.3 are double bond carbon signals at

positions

8 and 9; delta 66.2 is the oxygen carbon signal at the 7-position. According to the main spectral characteristics and by combining with the reference result, the compound is identified as ganoderic acid D (translated into ganoderic acid D, GA-D for short) with the chemical formula C₃₀H₄₂O₇Molecular weight 514, its structural formula is shown in formula I.

Example 3: anti-aging effect of GA-D natural compound

In order to show that the compound of the invention has the effect of delaying senescence of human mesenchymal stem cells, corresponding tests are carried out:

after pre-treating human mesenchymal stem cells with different concentrations (0.001. mu.M, 0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M, 100. mu.M) of GA-D, respectively, for 6H, H was added to the cells to a final concentration of 200. mu.M₂O₂After 2h and 48h of treatment, the generation of beta-galactosidase, a aging characteristic marker, was detected, and the results are shown in FIG. 3, and the inhibition of the production of beta-galactosidase and Reactive Oxygen Species (ROS) by GA-D was also detected, and the results are shown in FIG. 6. Compared with the normal group, the number of beta-galactosidase positive cells was significantly increased in the model group without the addition of GA-D treatment, and as shown in A in FIG. 6, the positive cell rate was increased from 4.53. + -. 1.13% to 46.92. + -. 0.74%. Compared with the model group, the administration group treated by GA-D with the concentration range of 0.1-10 mu M can obviously inhibit beta-an increase in galactosidase positive cells with an inhibition of 15.39% at 0.1 μ M for β -galactosidase positive cells; the inhibition rate of beta-galactosidase positive cells at 1. mu.M reached 40.3%, and especially the inhibition rate of beta-galactosidase positive cells at 10. mu.M reached 58.27%, as shown in B of FIG. 6. Therefore, the GA-D with the concentration of 1-10 mu M is adopted, and the effect is best; furthermore, GA-D inhibited the production of ROS in human mesenchymal stem cells, and the results are shown in C in FIG. 6. Subsequently, the expression level of p21 protein and the change in the total number of viable cells after GA-D pretreatment and 72h culture were further examined. As a result, the expression of p21 protein was significantly up-regulated in the model group, whereas the expression of p21 protein was significantly reduced by GA-D treatment, and the total number of viable cells was increased by 1.83 times compared to the model group, as shown in A of FIG. 7. These results suggest that GA-D significantly ameliorates the phenomenon of cell proliferation arrest caused by aging. In addition, the effect of GA-D on telomerase, a direct association with senescence, was also examined. As shown at B in FIG. 7, at H₂O₂In the model group, the telomerase content in cells is obviously reduced compared with that in the normal group, and after GA-D pretreatment, the telomerase content can be up-regulated. Indicating that the anti-senescence effect of GA-D is involved in telomerase activation.

The tests show that the GA-D compound has the effect of delaying senescence of the human mesenchymal stem cells, particularly obviously inhibits the increase of beta-galactosidase positive cells, inhibits the generation of ROS in the human mesenchymal stem cells, and can obviously reduce the expression of p21 protein. Therefore, the composition can be used for preparing the medicine for delaying senility, and can be prepared into different types of medicines such as tablets, capsules, pills, fat emulsion or ointment with medically acceptable pharmaceutical excipients for different people to use.

Example 4: effect of GA-D Natural Compounds on the growth of human mesenchymal Stem cells

In order to illustrate the safety of use of the compounds of the present invention, i.e., whether it is safe for human mesenchymal stem cells, a corresponding test was performed:

the GA-D with different concentrations (0.001 mu M, 0.01 mu M, 0.1 mu M, 1 mu M, 10 mu M and 100 mu M) is respectively used for pre-treating the human mesenchymal stem cells for 8 hours, then the complete culture medium is replaced, the cytotoxicity condition is detected by using the MTT method after 72 hours, the test result is shown in a histogram shown in figure 8, the cell viability is not different from that of a control group after the GA-D treatment, and the fact that the GA-D with different concentrations has no influence on the human mesenchymal stem cells, does not generate cytotoxicity and is safe is suggested.

The above-mentioned examples only express the specific embodiments of the present invention, but should not be interpreted as limiting the scope of the present 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.

Claims

1. Application of compound for delaying human mesenchymal stem cell senescence in preparation of drug for delaying stem cell senescence, and is characterized in that molecular formula of compound is C₃₀H₄₂O₇The structural formula is shown as the general formula (I):

the preparation method of the compound comprises the following steps: drying Ganoderma fruiting body, pulverizing into 200-mesh fine powder of 300 meshes, extracting with 80-95% methanol for 3 times, collecting supernatant, concentrating under reduced pressure to obtain extract concentrate, drying the concentrate to obtain extract, performing silica gel column chromatography, performing gradient elution with chloroform-methanol, collecting eluate with mobile phase of 70:30, separating the eluate with silica gel column and ODS open column, and performing reverse phase semi-preparative HPLC to obtain extract with chemical formula C₃₀H₄₂O₇The chemical structural formula of the compound is shown as the general formula (I).

2. The use according to claim 1, wherein the compound is administered to the medicament at a dose concentration of 0.001 to 100 μ M.

3. The application of claim 1, which is characterized in that the compound is prepared into a tablet, a capsule, a pill, a fat emulsion or an ointment with pharmaceutically acceptable pharmaceutical excipients, and the dosage concentration of the compound in the prepared medicine is 1-10 mu M.