CN116832047B - Application of ginsenoside Rg1 and derivatives thereof in vitro antagonism of replicative senescence of sub-totipotent stem cells - Google Patents
Application of ginsenoside Rg1 and derivatives thereof in vitro antagonism of replicative senescence of sub-totipotent stem cells Download PDFInfo
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- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention provides application of ginsenoside Rg1 and derivatives thereof in antagonizing replicative senescence of sub-totipotent stem cells in vitro or preparing biological products for antagonizing replicative senescence of sub-totipotent stem cells in vitro. Compared with the prior art, the invention has the following advantages: the ginsenoside Rg1 can inhibit the aging-related secretion phenotype of the replicative aging sub-totipotent stem cells, promote the activity of the replicative aging sub-totipotent stem cells, enhance the proliferation capacity of the replicative aging sub-totipotent stem cells, inhibit the apoptosis of the replicative aging sub-totipotent stem cells, enhance the osteogenic/adipogenic differentiation of the replicative aging sub-totipotent stem cells and promote the calcium ion flow of the replicative aging sub-totipotent stem cells, is vital for improving the aging sub-totipotent stem cells, and provides higher reference value for in-vivo experiments of anti-aging individuals.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of ginsenoside Rg1 and derivatives thereof in antagonizing the replicative senescence of sub-totipotent stem cells in vitro or preparing biological products for antagonizing the replicative senescence of the sub-totipotent stem cells in vitro.
Background
Stem cells are a type of cells which are not sufficiently differentiated, have high self-renewal capacity and can regenerate various tissues and organs, so that the stem cells have wide development prospects in research and application of tissue engineering and regenerative medicine. For mammals, stem cells can be classified into embryonic stem cells, which are derived from the intra-blastula cell mass, and adult stem cells, which are derived from various tissues. As science and technology advances, stem cell research has become more and more interesting, and they are of great importance in the field of biology. Sub-totipotent stem cells and mesenchymal stem cells are the two most common types of stem cells, which differ in morphology and function.
The sub-totipotent stem cells are important adult stem cells, exist in the interstitium of various tissues and organs, especially bone marrow and subcutaneous fat, have multidirectional differentiation potential, and can be differentiated into osteoblasts, adipocytes, chondrocytes, nerve cells and the like. In research and application of tissue engineering and regenerative medicine, the sub-totipotent stem cells become an important seed cell due to the characteristics of wide sources, low immunogenicity, various differentiation potential, no ethical disputes and the like. The sub-totipotent stem cell is a stem cell which grows in a 'fibroblast-like' adherence way and expresses various cell surface markers when being cultured in vitro. However, as with other normal somatic cells, sub-totipotent stem cells have limited in vitro expansion algebra and enter a replicative aging state after a certain number of mitosis, and are specifically characterized in that: the method has the advantages of large volume, reduced cell activity, reduced proliferation and differentiation capacity, overload of mitochondrial calcium ions and the like, and brings difficulty to the fields of scientific research, tissue engineering, regenerative medicine application and the like of the sub-totipotent stem cells, so that it is important to explore how to improve the in vitro replicative senescence of the sub-totipotent stem cells.
Ginsenoside is the most main active component in traditional Chinese medicine ginseng, and belongs to steroid compounds. The basic structure of ginsenoside is tetracyclic sterane steroid nucleus formed by 30 carbon atoms, and can be classified into dammarane type (such as ginsenoside Rb1, rb2, rb3, rc, rd, re, rg1, rg2, rg3, rh2, etc.) and oleanane type due to different numbers, positions and types of hydroxyl groups and glycosyl groups of C-3 and C-6 in the skeleton structure of the mother nucleus. In recent years, part of ginsenoside has been reported to be anti-aging, inflammation-regulating, anti-oxidation, anticancer and the like through in vivo and in vitro experiments. Ginsenoside Rg1 (Ginsenoside Rg1, molecular formula C) 42 H 72 O 14 ) The ginsenoside has a large content, is one of the most effective components of ginseng roots, has various pharmacological activities, and has been reported to play a role in protecting tissues and organs such as nerves, livers and kidneys by promoting cell proliferation, reducing oxygen free radical generation, inhibiting inflammatory factor secretion and other mechanisms, so that the ginsenoside has great potential in treating metabolic syndrome, alzheimer's disease, aging resistance and the like. However, whether ginsenoside Rg1 can improve the replicative senescence of human tissue-derived sub-totipotent stem cells in vitro has not been reported yet.
Disclosure of Invention
Aiming at the technical limitations, the invention provides application of ginsenoside Rg1 and derivatives thereof in-vitro antagonizing the replicative senescence of the sub-totipotent stem cells or preparing biological products for in-vitro antagonizing the replicative senescence of the sub-totipotent stem cells; which overcomes the deficiencies and drawbacks mentioned in the background.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides application of ginsenoside Rg1 and derivatives thereof in regulating activity of sub-totipotent stem cells or preparing biological products for regulating activity of sub-totipotent stem cells, wherein the application is as follows: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of cell viability of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of cell viability of replicative senescent sub-totipotent stem cells.
Optionally, the above application may further be specifically: application of ginsenoside Rg1 and its derivatives in vitro antagonizing replicative senescence of sub-totipotent stem cells or preparing biological products for in vitro antagonizing replicative senescence of sub-totipotent stem cells.
Optionally, the above application may further be specifically: the application of ginsenoside Rg1 and derivatives thereof in inhibiting the aging-related secretion phenotype of replicative aging sub-totipotent stem cells in vitro or preparing biological products for inhibiting the aging-related secretion phenotype of replicative aging sub-totipotent stem cells in vitro.
Optionally, the above application may further be specifically: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of cell proliferation of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of cell proliferation of replicative senescent sub-totipotent stem cells.
Optionally, the above application may further be specifically: the application of ginsenoside Rg1 and derivatives thereof in inhibiting apoptosis of replicative senescent sub-totipotent stem cells in vitro or preparing biological products for inhibiting apoptosis of replicative senescent sub-totipotent stem cells in vitro.
Optionally, the above application may further be specifically: the application of ginsenoside Rg1 and derivatives thereof in vitro promotion of osteogenic differentiation/adipogenic differentiation of replicative senescent sub-totipotent stem cells or preparation of biological products for in vitro promotion of osteogenic differentiation/adipogenic differentiation of replicative senescent sub-totipotent stem cells.
Optionally, the above application may further be specifically: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of the calcium ion flow of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of the calcium ion flow of replicative senescent sub-totipotent stem cells.
The chemical structural formulas of ginsenoside Rg1 and its derivatives ginsenoside F1 and ginsenoside Rh1 are shown in figure 9.
Alternatively, for the above application, the effective concentration of ginsenoside Rg1 is 5-150. Mu.M, preferably 6.25-100. Mu.M, more preferably 50. Mu.M.
The effective concentration can be selected from 5. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 125. Mu.M, 150. Mu.M; the most effective concentration is 50. Mu.M.
Optionally, in the application, the purity of the ginsenoside Rg1 and the derivatives thereof is more than or equal to 98%.
Alternatively, for the above application, the biologic is a drug.
The following 3 patent publications related to the present patent application were found by searching:
1. use of ginsenoside 20 (R) -25-OH-Rg3 to promote proliferation of umbilical cord-derived sub-totipotent stem cells in vitro (CN 110055214A);
2. application of ginsenoside Rg2 in promoting mesenchymal stem cell proliferation and inhibiting replicative senescence in vitro (CN 115261311A);
3. application of hollow vanadium dioxide core-membrane composite structure drug carrier modified by metal-organic ligand framework ZIF-67 (CN 114191413A).
By way of comparison, the present patent application is substantially different from the above 3 patent publications. Firstly, the chemical structures of the ginsenoside Rg1, the 20 (R) -25-OH-Rg3 and the ginsenoside Rg2 are different; secondly, the invention uses the human sub-totipotent stem cells, has the multi-directional differentiation potential and the immunoregulation function, and has more potential in further tissue engineering and regenerative medicine application; thirdly, the invention relates to the improvement effect of ginsenoside Rg1 on various characteristics of replicative senescence, but is not limited to proliferation level, has remarkable innovation in calcium ion flow, and provides a firmer foundation in further scientific research and medical application; fourth, the stem cells used in patent document 3 are HMSCs, i.e., human bone marrow stromal stem cells, which are different from the human sub-totipotent stem cells used in the present patent application.
Compared with the prior art, the invention has the following advantages:
1. the ginsenoside Rg1 can inhibit the aging-related secretion phenotype of the replicative aging sub-totipotent stem cells, is crucial for improving the aging sub-totipotent stem cells, and provides higher reference value for in-vivo experiments of anti-aging individuals.
2. The ginsenoside Rg1 can improve the cell activity of the replicative senescence sub-totipotent stem cells, enhance the proliferation capability of the replicative senescence sub-totipotent stem cells, inhibit the apoptosis of the replicative senescence sub-totipotent stem cells, and has important significance for in vitro culture and expansion of the sub-totipotent stem cells, tissue engineering and regenerative medicine and senescence research of the sub-totipotent stem cells.
3. The ginsenoside Rg1 can obviously enhance the osteogenic differentiation and adipogenic differentiation potential of replicative senescent sub-totipotent stem cells, and can bring important value for treating age-related diseases such as bone defect and the like.
4. The ginsenoside Rg1 can promote the flow of calcium ions in cytoplasm of replicative aging sub-totipotent stem cells, and brings important thought for the innovative scientific research related to the activation of stem cells by traditional Chinese medicine monomers through electrophysiology.
Drawings
FIG. 1 shows the results of an IL-1βElisa assay (IL-1β concentration and mRNA expression level) for the in vitro culture of human replicative senescent pluripotent stem cells with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the invention.
FIG. 2 shows the results of an IL-6Elisa assay (IL-6 concentration and mRNA expression level) for the in vitro culture of human replicative senescent sub-totipotent stem cells with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 3 shows the results of experiments for detecting the viability of CCK8 cells obtained by culturing human replicative senescent sub-totipotent stem cells in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 4 shows the results of a multiplication experiment of cell population of human replicative senescent sub-totipotent stem cells cultured in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 5 shows the results of apoptosis test of human replicative senescent sub-totipotent stem cells cultured in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 6 shows the results of adipogenic differentiation induction experiments of human replicative senescent sub-totipotent stem cells cultured in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 7 shows the results of an osteogenic differentiation induction experiment of human replicative senescent sub-totipotent stem cells cultured in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 8 shows the results of the experiments for detecting the concentration of calcium ions of the human replicative senescent sub-totipotent stem cells cultured in vitro with ginsenoside Rg1, F1, rh1, rg3 according to an embodiment of the present invention.
FIG. 9 shows chemical structural formulas of ginsenoside Rg1, ginsenoside F1, ginsenoside Rh1 and ginsenoside Rg3 according to an embodiment of the present invention.
Fig. 10 is a structural transformation flow of ginsenoside Rg1 and its derivatives ginsenoside F1 and ginsenoside Rh1 in an embodiment of the present invention.
FIG. 11 shows the effect of ginsenoside Rh1 on SH-SY5Y cells according to an embodiment of the present invention; wherein (a) is ginsenoside Rh1 used for 24 hours at a concentration of 6.25, 12.5, 25, 50 and 100. Mu.M; (b) For inducing neuronal cell death in SH-SY5Y cells in a dose-dependent manner for aβ oligomers, SH-SY5Y cells were treated with different concentrations of aβ oligomers for 24 hours, <0.01 for p compared to control (Con); (c) Inhibition of aβ oligomer-induced neuronal cell death in SH-SY5Y cells for ginsenoside Rh1 was p <0.001 compared to control (Con), p <0.05, p <0.01 compared to aβ oligomer (aβ).
FIG. 12 shows the results of Abeta oligomers inhibiting Akt and GSK-3 pathway and activating p38 MAPK pathway in SH-SY5Y cells according to an embodiment of the present invention; wherein, (a) is the detection result of phosphorylated Akt (Ser 473); (b) is the detection result of phosphorylated GSK-3 beta (Ser 9); (c) Is the detection result of phosphorylation-p 38 MAPK (Thr 180/Tyr 182); all data are expressed as mean ± SE, the experiment was performed at least three times, p <0.01, p <0.05 and p <0.001 compared to the control group (Con).
FIG. 13 shows the chemical reaction scheme of the PEG derivatization process of ginsenoside Rg1 in an embodiment of the present invention.
Fig. 14 shows the results of examining the stability of ginsenoside Rg1, ginsenoside derivative PEG-Rg1, and physical mixture of ginsenoside Rg1 and PEG in rat stereoscopic in vitro stomach (n=3), wherein a is ginsenoside Rg1, B is ginsenoside derivative PEG-Rg1, and C is physical mixture of ginsenoside Rg1 and PEG in an embodiment of the present invention.
FIG. 15 shows a cell model of a sub-totipotent stem cell capable of expressing pluripotent genes Myc, klf4 and Gmnn higher than embryonic stem cells according to an embodiment of the invention; similar to embryonic stem cells, sub-totipotent stem cells exhibit a normochromic state by electron microscopy of the nuclei of high pressure frozen cells. The pluripotent, reproductive layer and differentiation related genes of the sub-totipotent stem cells such as lysine No. 4 and/or lysine No. 27 of histone H3 near Myc, oct4, sall4, sox2, klf4, nanog and the like are subjected to a trimethylation modification, so that the sub-totipotent stem cells have epigenetic pluripotency.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.
The present embodiments are intended to be illustrative, not restrictive, and not to limit the scope of the invention;
the raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention, unless otherwise specified, are all conventional in the art;
all data statistical analyses were performed using GraphPad Prism 9 (GraphPad Prism, san Diego, CA) software and statistical comparisons were performed between the two groups using t-test. The comparison between the groups adopts single-factor analysis of variance. The differences are statistically significant, P <0.05, < P <0.01, < P <0.001, < P <0.0001, respectively.
For a further understanding of the present invention, the present invention will be described in further detail with reference to the following preferred embodiments.
Example 1
The application of ginsenoside Rg1 and derivatives thereof in regulating the activity of the sub-totipotent stem cells or preparing biological products for regulating the activity of the sub-totipotent stem cells is as follows: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of cell viability of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of cell viability of replicative senescent sub-totipotent stem cells.
The method can also specifically be as follows: application of ginsenoside Rg1 and its derivatives in vitro antagonizing replicative senescence of sub-totipotent stem cells or preparing biological products for in vitro antagonizing replicative senescence of sub-totipotent stem cells.
The method can also specifically be as follows: the application of ginsenoside Rg1 and derivatives thereof in inhibiting the aging-related secretion phenotype of replicative aging sub-totipotent stem cells in vitro or preparing biological products for inhibiting the aging-related secretion phenotype of replicative aging sub-totipotent stem cells in vitro.
The method can also specifically be as follows: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of cell proliferation of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of cell proliferation of replicative senescent sub-totipotent stem cells.
The method can also specifically be as follows: the application of ginsenoside Rg1 and derivatives thereof in inhibiting apoptosis of replicative senescent sub-totipotent stem cells in vitro or preparing biological products for inhibiting apoptosis of replicative senescent sub-totipotent stem cells in vitro.
The method can also specifically be as follows: the application of ginsenoside Rg1 and derivatives thereof in vitro promotion of osteogenic differentiation/adipogenic differentiation of replicative senescent sub-totipotent stem cells or preparation of biological products for in vitro promotion of osteogenic differentiation/adipogenic differentiation of replicative senescent sub-totipotent stem cells.
The method can also specifically be as follows: the application of ginsenoside Rg1 and derivatives thereof in-vitro promotion of the calcium ion flow of replicative senescent sub-totipotent stem cells or preparation of biological products for in-vitro promotion of the calcium ion flow of replicative senescent sub-totipotent stem cells.
The chemical structural formulas of ginsenoside Rg1 and its derivatives ginsenoside F1 and ginsenoside Rh1 are shown in figure 9.
The effective concentration of ginsenoside Rg1 is 5-150. Mu.M, preferably 6.25-100. Mu.M, and more preferably 50. Mu.M.
The effective concentration can be selected from 5. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 125. Mu.M, 150. Mu.M; the most effective concentration is 50. Mu.M.
The purity of the ginsenoside Rg1 and the derivatives thereof is more than or equal to 98 percent.
The biological product is a medicine.
Sub-totipotent stem cells are a common type of stem cell that can differentiate into different cell types, which can be transformed into neural cells, cardiomyocytes, hepatocytes, etc. They are characterized by a complete morphology, a strong regeneration capacity, and a long-term metabolism, thus keeping the cell system healthy.
Mesenchymal stem cells are a type of differentiable cells, which are a type of multifunctional cells that can differentiate into various cell types, such as vascular cells, blood cells, hepatocytes, cardiomyocytes, etc. They are characterized by incomplete morphology, strong regeneration capacity, and maintenance of long-term metabolism, thereby keeping the cell system healthy.
The distinction between sub-totipotent stem cells and mesenchymal stem cells is mainly reflected in morphology and function. The sub-totipotent stem cells have complete morphology and can be differentiated into various cells such as nerve cells, cardiac muscle cells, liver cells and the like, while the mesenchymal stem cells have incomplete morphology and can be differentiated into only one cell such as vascular cells, blood cells, liver cells, cardiac muscle cells and the like. In addition, the regeneration capability of the sub-totipotent stem cells is stronger, long-term metabolism can be maintained, and the regeneration capability of the mesenchymal stem cells is weaker, so that the metabolism can be maintained only in a short period.
Compared with common adult multipotent stem cells such as nerve stem cells, hair follicle stem cells and the like, the sub-multipotent stem cells have multidirectional differentiation potential, namely, can directionally differentiate into cells of multiple germ layers such as osteoblasts, chondrocytes, adipocytes, hepatocytes, glial cells, myocytes, endothelial cells and the like. The applicant originally confirms the multi-directional differentiation potential of the sub-totipotent stem cells through the early solid research foundation, and specifically reveals the molecular characteristics of the sub-totipotent stem cells, which are different from the common stem cells, through the technologies of flow cytometry, single cell sequencing and the like.
The sub-totipotent stem cells highly express multipotent genes Myc, klf4 and Gmnn. Similar to embryonic stem Cells, sub-totipotent stem Cells exhibited a normative chromatin state by electron microscopy analysis of nuclei of high pressure frozen Cells, and the genes related to pluripotency, reproductive specifications and differentiation were modified by H3K4me3 or by H3K4me3 and H3K27me3 together, which indicated that sub-totipotent stem Cells had epigenetic pluripotency, as shown in FIG. 15 (Li H, zhu L, chen H, li T, han Q, wang S, yao X, feng H, fan L, gao S, boyd R, cao X, zhu P, li J, keating A, su X, zhaoRC. Generation of Functional Hepatocytes from Human Adipose-development MYC+KLF4+GMNN+ Stem Cells Analyzed by Single-RNA-Seq Profile. Vector media 2018Nov;7 (11) 792-805.Doi: 10.1002/tm. 3517-0273.Epub2018Sep 11.PMID:30272835;PMCID:PMC6216430. FIG. 15 shows that the pluripotent stem cells highly express the multipotent genes Myc, klf4 and Gmnn, and that the nuclei are in a more open euchromatin state, and that the multipotent, germ layer specification and differentiation-related genes such as lysine No. 4 and/or lysine No. 27 of histone H3 near Myc, oct4, sall4, sox2, klf4, nanog and the like are subjected to trimethylmodification, compared with the embryonic stem cells, so that the pluripotent stem cells have epigenetic multipotency.
In addition, the sub-totipotent stem cells have homing repair function, namely under the induction of the micro environment of the tissue organ at the damaged part, the homing sub-totipotent stem cells divide, proliferate, directionally differentiate and integrate into the damaged tissue organ to repair or regenerate the diseased tissue organ. The characteristics show that the sub-totipotent stem cells are important stem cells with research and transformation values no matter the important scientific problem of aging mechanism in life process or the clinical application in the field of stem cell regeneration medicine. Therefore, compared with common stem cells, the anti-aging research of the sub-totipotent stem cells is very important for the anti-aging of the whole life body and the in-vitro preparation and clinical application of the sub-totipotent stem cells.
Example 2
Human sub-totipotent stem cell culture:
taking P0 generation human sub-totipotent stem cells, using complete culture medium (DMEM/F12+2% fetal bovine serum+penicillin 100 IU/mL+streptomycin 100 μg/mL) at 37deg.C, 5% CO 2 Culturing in incubator, and changing culture medium every 2 days. When the cell growth density reaches 80% -90%, the 18 th-20 th generation human sub-totipotent stem cells are used for each experiment.
Example 3
Detection of aging secretion phenotype of human sub-totipotent stem cells:
the 18 th-20 th generation human sub-totipotent stem cells are used for aging secretion phenotype detection experiments, and the detection indexes of the experiments are inflammatory factors interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6). The experiment is divided into a blank group and a ginsenoside Rg1 administration group, and a ginsenoside Rg3 administration group. Ginsenoside Rg1, F1, rh1 and Rg3 powder is dissolved, filtered, sterilized and added into a complete culture medium for standby. Generation 18-20 human sub-totipotent stem cells are expressed as 1X 10 5 The density of each hole is inoculated in a 6-hole plate, after the cell adhesion condition is good, the old culture medium is discarded, and the complete culture medium corresponding to the experimental group is respectively added into the six-hole plate. 72 smallAnd respectively collecting cell supernatants of each hole, detecting the concentration of IL-1 beta and IL-6 according to the steps provided by the specification of an Elisa kit, respectively collecting adherent cells in each hole, and detecting the relative mRNA expression level of IL-1 beta and IL-6 according to the steps of general RNA extraction and real-time fluorescence quantitative PCR.
The experimental results are shown in fig. 1 and 2. FIG. 1 shows IL-1β measurement results, and to the left, elisa measurement results, namely, the secretion of IL-1β by aging cells in Rg1, F1, rh 1-administered groups was reduced compared with the blank group, wherein the secretion of IL-1β by 50. Mu.M Rg 1-administered group was relatively reduced by 20.5% + -2.4% and significantly lower than that by Rg3; the right side is the real-time fluorescence quantitative PCR detection result, namely, compared with a blank group, the relative mRNA expression level of the aging cells IL-1 beta of the Rg1, F1 and Rh1 administration group is reduced, wherein the relative mRNA expression of the IL-1 beta of the 50 mu M Rg1 administration group is relatively reduced by 23% +/-4.6%, and is obviously lower than that of Rg3. FIG. 2 shows IL-6 detection results, and to the left, elisa detection results, namely, the secretion of IL-6 by aging cells in Rg1, F1 and Rh 1-administered groups is reduced compared with that in a blank group, wherein the secretion of IL-6 in a 50 mu M Rg 1-administered group is relatively reduced by 21.6% + -1.6% and is significantly lower than that in Rg3; the right side is the real-time fluorescence quantitative PCR detection result, namely, compared with a blank group, the relative mRNA expression level of the aging cells IL-6 of the Rg1, F1 and Rh1 administration group is reduced, wherein the relative mRNA expression level of the aging cells IL-6 of the Rg3 administration group is not significantly different, and the relative mRNA expression of the IL-6 of the Rg1 administration group is reduced by 22.8+/-2.1 percent, namely, the relative mRNA expression level is superior to that of the Rg3 administration group.
Example 4
Human sub-totipotent stem cell CCK8 cell viability detection:
the 18 th-20 th generation human sub-totipotent stem cells are used for CCK8 cell viability detection experiments. Experimental group example 3 was completely identical. The 18 th-20 th generation human sub-totipotent stem cells are inoculated into 96-well plates at a density of 5000 cells/well, 6 compound wells are formed in each group, and after the cell adhesion condition is good, old culture medium is discarded, and new medicine-containing/medicine-free culture medium is replaced. After 72 hours old medium was discarded, and after adding CCK 8-containing solution according to the procedure provided in the CCK8 kit instructions, the solution was incubated at 37℃for 4 hours, and the absorbance of each well was measured by using a microplate reader at 490 nm.
The experimental results are shown in FIG. 3. FIG. 3 shows the absorbance detection results of CCK8, and the viability of the aging cells of the Rg1, F1 and Rh1 administration group is improved compared with that of the blank group, wherein the viability of the cells of the Rg1 administration group with 50 mu M is improved by 120.2+/-11.3% relatively, and the cell viability of the cells of the Rg1 administration group with 50 mu M is obviously better than that of the cells of the Rg3 administration group with the Rg 1.
Example 5
Human sub-totipotent stem cell population doubling time detection:
the 18 th-20 th generation human sub-totipotent stem cells are used for cell population proliferation multiplication experiments. The experimental group and the ginsenoside Rg1 concentration are completely consistent with example 3. Inoculating 18-20 th generation human sub-totipotent stem cells into 96-well plate at 5000 times/well, 9 multiple wells each, discarding old culture medium after cell adhesion is good, and taking 3 multiple Kong Yi enzymes each to digest in cell counter for counting (i.e. cell number before drug treatment, N) 0 ) The rest is replaced with new medicated/non-medicated medium. After 72 hours of medium removal, cells from each well were trypsinized and counted in a cytometer (i.e., the number of cells after drug treatment, nt), and cell population doubling time = t×lg2/lg (Nt/N) 0 ) Wherein T is 72.
The experimental results are shown in FIG. 4. FIG. 4 shows the results of the population doubling time test, wherein the population doubling time of the aging cells of the Rg1, F1 and Rh1 administration group is shortened compared with that of the blank group, and the population doubling time of the 50 mu M Rg1 administration group is relatively shortened by 31.6+/-1.1%, and is remarkably superior to that of the Rg3 administration group.
Example 6
Detection of apoptosis of human sub-totipotent stem cells:
the 18 th-20 th generation human sub-totipotent stem cells are used for apoptosis experiments. The experimental group and the ginsenoside Rg1 concentration are completely consistent with example 3. Generation 18-20 human sub-totipotent stem cells are expressed as 1X 10 5 The density of each hole is inoculated in a 6-hole plate, and after the cell adhesion condition is good, the old culture medium is discarded, and the new medicine-containing/medicine-free culture medium is replaced. After 72 hours, detection was performed according to the procedure provided in the Annexin V-FITC/PI apoptosis detection kit instructions, the principle being as follows: in normal living cells, phosphatidylserine (PS) is located inside the cell membrane, but in early apoptotic cells PS flips from inside to the surface of the cell membrane, bound by Annexin-v with high affinity; iodination ofPropidium (PI) is a nucleic acid dye that does not penetrate the intact cell membrane of normal or early apoptotic cells, but can stain the nucleus of cells red through the cell membrane of late apoptotic and necrotic cells to distinguish between surviving early cells and necrotic or late apoptotic cells. Thus, annexin V was used in combination with PI and examined by flow cytometry, the most numerous of the late apoptotic cell populations exhibited by replicative senescence were located to the right of the axis.
The experimental results are shown in FIG. 5. FIG. 5 shows the results of flow cytometry detection of apoptotic cells, with the sum of right side (Q1-UR+Q1-LR) as the ratio of late apoptotic cells, the ratio of late apoptotic cells of aging cells of Rg1, F1, rh 1-dosed groups was significantly reduced compared to the blank group, wherein 50. Mu.M of late apoptotic cells of Rg 1-dosed group was relatively reduced by 36.8%, and was superior to Rg 3-dosed group.
Example 7
And (3) detecting the adipogenic differentiation of the human sub-totipotent stem cells:
the 18 th-20 th generation human sub-totipotent stem cells are used for adipogenic differentiation induction experiments. The experimental group and the ginsenoside Rg1 concentration are completely consistent with example 3. Generation 18-20 human sub-totipotent stem cells are expressed as 2X 10 4 The density of each hole is inoculated in a 24-hole plate, after the cell adhesion condition is good, the old culture medium is abandoned, the new medicine-containing/medicine-free adipogenic induction culture medium is replaced, the liquid is replaced every 3 days, the intracellular lipid drops, namely the fat cell markers, are dyed according to the general step of oil red O dyeing on the 12 th day, and the number and the size of the lipid drops can be used for representing the differentiation efficiency of the human sub-totipotent stem cells to the fat cells. Photographing under a microscope after dyeing is finished, adding isopropanol extraction oil red O dye after photographing is finished, and measuring the absorbance value of each hole by using a microplate reader at 570nm wavelength to perform semi-quantitative analysis.
The experimental results are shown in FIG. 6. FIG. 6 shows the results of lipid-forming differentiation test of senescent cells, and the upper graph shows the staining condition of lipid droplets of each group under a microscope, wherein compared with a blank group, the lipid-forming differentiation efficiency of senescent cells in Rg1, F1 and Rh1 administration groups is obviously improved, and the lipid-forming differentiation efficiency of senescent cells in Rg3 administration group is also improved; the lower panel shows the results of semi-quantitative analysis of lipid droplets in each group, and the lipid-forming differentiation efficiency of senescent cells in the 50. Mu.M Rg 1-dosed group is improved by 66.6% + -16.9% compared with that in the blank group, and is superior to that in the Rg 3-dosed group.
Example 8
Human sub-totipotent stem cell osteogenic differentiation assay:
the 18 th-20 th generation human sub-totipotent stem cells are used for osteogenic differentiation induction experiments. The experimental group and the ginsenoside Rg1 concentration are completely consistent with example 3. Generation 18-20 human sub-totipotent stem cells are expressed as 2X 10 4 The density of each hole is inoculated in a 24-hole plate, after the cell adhesion condition is good, the old culture medium is abandoned, the new medicine-containing/medicine-free osteogenesis induction culture medium is replaced, the liquid is changed every 3 days, the deposited calcium salt in the cell is dyed according to the general step of alizarin red dyeing on the 12 th day, the deposited calcium salt is an osteogenesis cell marker, the dyeing degree can be used for representing the differentiation efficiency of the human sub-totipotent stem cells to the osteogenesis cells, and the osteogenesis cells are photographed under a microscope after the dyeing is completed.
The experimental results are shown in FIG. 7. FIG. 7 shows the results of the osteoblast differentiation test, and the deposition of calcium salt in each group was observed under a microscope, and compared with the blank group, the osteoblast differentiation efficiency of the aging cells in the Rg1, F1 and Rh1 administration group was significantly improved, and the osteogenic differentiation efficiency of the aging cells in the Rg1 administration group was 50. Mu.M better than that in the Rg3 administration group.
Example 9
Calcium ion detection of human sub-totipotent stem cells:
the 18 th-20 th generation human sub-totipotent stem cells are used for calcium ion detection experiments. The experimental group and the ginsenoside Rg1 concentration are completely consistent with example 3. Generation 18-20 human sub-totipotent stem cells are expressed as 5×10 4 The density of each hole is inoculated into a 12-hole plate, and after the cell adhesion condition is good, the old culture medium is discarded, and the new complete culture medium containing or not containing the medicine is replaced. After 72 hours, rhod-2-AM kit and Calbryte were used TM The 520AM kit provides the procedure for detection. Rhod-2-AM is a mitochondrial specific calcium ion probe, and can be quantitatively detected by a multifunctional enzyme-labeled instrument (590 nm) after being combined with calcium ions. Calbryte TM 520AM is a cytoplasmic calcium ion probe, and can be quantitatively detected by a flow cytometer (FITC channel) after being combined with calcium ions.
The experimental results are shown in FIG. 8. The left side is the mitochondrial calcium ion detection result, namely compared with a blank group, the mitochondrial calcium ion overload condition of the aging cells of the Rg1, F1 and Rh1 administration group is obviously improved, the mitochondrial calcium ion concentration is relatively reduced, wherein the mitochondrial calcium ion concentration of the aging cells of the Rg1 administration group with 50 mu M is relatively reduced by 38.4+/-3 percent, and the mitochondrial calcium ion concentration is superior to that of the Rg3 administration group; the right side is the result of the cytoplasmic calcium ion detection, namely, compared with a blank group, the concentration of the cytoplasmic calcium ion of the aging cells of the Rg1, F1 and Rh1 administration group is obviously increased, wherein the concentration of the cytoplasmic calcium ion of the aging cells of the 50 mu MRg1 administration group is relatively increased by 27.3+/-3.1 percent, which is superior to that of the Rg3 administration group.
Calcium ion is one of the important second messengers that mediate cell signaling. The inside of the cell is usually used as a 'calcium ion library' by two cell organelles of an endoplasmic reticulum and a mitochondria, and calcium ions are stored to regulate the overall calcium ion balance of the cell. In the case of aging cells, the mitochondria excessively store calcium ions, which causes calcium ion overload, which is not only detrimental to the mitochondria itself and the whole cell, but also affects the signal transduction and communication of the cells, thereby exacerbating the aging of the cells and their microenvironment. Ginsenoside Rg1 and its derivatives can release calcium ions overloaded by mitochondria into cytoplasm, i.e. reduce mitochondrial calcium ion concentration and improve cytoplasmic calcium ion concentration, which is beneficial to inhibiting apoptosis of aging cells, reducing secretion of inflammatory factors of aging cells, etc., thereby exerting antagonistic replicative aging of sub-totipotent stem cells.
Example 10
(1) The intestinal desugarized metabolites of ginsenoside Rg1 in vivo include ginsenoside Rh1, ginsenoside F1 and protopanaxatriol (PPT), the chemical structures of which are shown in FIG. 10 (Wu JJ, yang Y, wan Y, xia J, xu JF, zhang L, liu D, chen L, tang F, ao H, peng C.New insights into the role and mechanisms of ginsenoside Rg1 in the management of Alzheimer's disease.biomed pharmacothers.2022 Aug; 152:113207.Doi:10.1016/j.biosphaha.2022.113207.Epub 2022Jun 3.PMID:35667236.). The study found that ginsenoside Rh1, for example, among these derivatives, acts mainly by decreasing β -amyloid production, increasing acetylcholine levels, antioxidant and anti-apoptotic to combat Alzheimer's disease (age-related aging-related disease), the main mechanism including modulation of PI3K/AKT and BDNF signaling pathways, and the related data are shown in FIGS. 11 and 12 (M.park, S. -H.Kim, H. -J.Lee.Ginsenoside Rh1 exerts neuroprotective effects by activating the PI K/Akt pathway in Amyloid- β reduced SH-SY5Y cells, appl.Sci.,11 (2021), p.5654, doi.org/10.3390/app 11125654). FIG. 11 shows that SH-SY5Y cells were treated with Rh1 (6.25. Mu.M to 100. Mu.M) at various concentrations for 24 hours, and cytotoxicity was not detected in SH-SY5Y cells, as in FIG. 11 (a); next, the concentration of aβ was screened by treating cells with different concentrations of aβ oligomer (0.25 to 4 μm) for 24 hours, and it was found that aβ was able to induce cytotoxicity in a concentration-dependent manner, as shown in fig. 11 (b); in addition, in order to determine the neuroprotective effect of ginsenoside Rh1, SH-SY5Y cells were treated with A.beta.1. Mu.M and various concentrations of ginsenoside Rh1 (5, 10, 20 and 40. Mu.M) for 24 hours, and cell viability analysis showed that ginsenoside Rh1 was able to inhibit A.beta.induced SH-SY5Y cell death, as shown in FIG. 11 (c). FIG. 12 shows that 1. Mu.M of Abeta oligomer was exposed to SH-SY5Y cells for various durations and that expression levels of phosphorylated Akt/Akt, phosphorylated GSK-3 beta/GSK-3 beta, and phosphorylated p38 MAPK/p38 MAPK were observed; it was found that 1. Mu.M of Abeta was able to down-regulate both phospho-Akt/Akt and phospho-GSK-3. Beta. (FIGS. 12a, b) and up-regulate phospho-p 38 MAPK/p38 MAPK in a time-dependent manner (FIG. 12 c); this suggests a role for the PI3K/Akt pathway in aβ -induced cytotoxicity of SH-SY5Y cells, where aβ is able to down-regulate phosphorylated Akt/Akt and phosphorylated GSK-3/GSK-3, as well as up-regulate phosphorylated p38 MAPK/p38 MAPK.
(2) Through the stability experiment of the PEG-Rg1 modified by ginsenoside Rg1 and polyethylene glycol (PEG) in the rat in-vitro stomach, the PEG-Rg1 can improve the problem of easy decomposition in the stomach in a free state, and greatly improve the stability of the medicament. No ginsenoside Rg1 was detected in the physiological saline solution, indicating that the sample did not exude to the physiological saline medium through the stomach wall, and the measured content in the gastric homogenate was the undissociated Rg1 content. The stability of Rg1 in the rat in-vitro stomach is poor, the Rg1 is measured to be 26.8% at 2h, and the degradation is 73.2%; the stability of the physical mixture of Rg1 and PEG in the rat in-vitro stomach is not significantly different from that of the original drug, and the Rg1 is 31.6 percent and the degradation is 68.4 percent when measured for 2 hours; the stability of PEG-Rg1 in the in-vitro stomach of the rat is improved, 81.8% of Rg1 is still detected in 2 hours, only 18.2% of PEG-Rg1 is degraded, and the result shows that the stability of PEG-Rg1 in the stomach is obviously improved. The relevant data are shown in FIGS. 13 and 14 (Liu Mei, wang Li, hu Kaili, etc. ginsenoside Rg1 PEG modification and stability experimental study, J.Chinese traditional medicine, 2012, 37 (10): 1378-1382.). The increase of the PEG-Rg1 of the polyethylene glycol derivative of the ginsenoside Rg1 in vivo stability lays a good foundation for the anti-aging in vivo/in vitro research of the ginsenoside Rg 1. Fig. 13 shows the chemical reaction formula of the PEG derivatization process of ginsenoside Rg1, and fig. 14 shows the results of investigation of the stability of Rg1, PEG-Rg1, a physical mixture of Rg1 and PEG in rat stereo-in-vitro stomach (n=3).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (9)
1. The application of the ginsenoside Rg1 and the derivatives thereof in preparing biological products for regulating the activity of the sub-totipotent stem cells is characterized in that the chemical formula of the ginsenoside Rg1 is shown in a formula (I), the derivatives of the ginsenoside Rg1 are ginsenoside F1 or ginsenoside Rh1, the chemical formula of the ginsenoside F1 is shown in a formula (II), and the chemical formula of the ginsenoside Rh1 is shown in a formula (III);
the application is specifically as follows: application of ginsenoside Rg1 and derivatives thereof in preparing biological products for promoting cell viability of replicative senescent sub-totipotent stem cells in vitro;
the effective concentration of the ginsenoside Rg1 is 50 mu M;
the preparation method of the sub-totipotent stem cells comprises the following steps: taking P0 generation human sub-totipotent stem cells, and using a complete culture medium at 37 ℃ and 5% CO 2 Culturing in incubator, changing culture medium every 2 days, and transferring to 18-20 th generation human sub-totipotent stem cells when the cell growth density reaches 80% -90%;
the complete culture medium comprises the following components: DMEM/F12+2% foetal calf serum+penicillin 100 IU/mL+streptomycin 100. Mu.g/mL.
2. The use according to claim 1, characterized in that it is in particular: the application of ginsenoside Rg1 and its derivatives in preparing biological products for in vitro antagonizing replicative senescence of sub-totipotent stem cells.
3. The use according to claim 1, characterized in that it is in particular: the application of ginsenoside Rg1 and derivatives thereof in preparing biological products for inhibiting aging-related secretion phenotype of replicative aging sub-totipotent stem cells in vitro.
4. The use according to claim 1, characterized in that it is in particular: the application of ginsenoside Rg1 and its derivatives in preparing biological product for promoting cell proliferation of replicative senescent sub-totipotent stem cells in vitro is provided.
5. The use according to claim 1, characterized in that it is in particular: the application of ginsenoside Rg1 and derivatives thereof in preparing biological products for inhibiting apoptosis of replicative senescent sub-totipotent stem cells in vitro.
6. The use according to claim 1, characterized in that it is in particular: application of ginsenoside Rg1 and its derivatives in preparing biological product for promoting osteogenic differentiation/adipogenic differentiation of replicative aging sub-totipotent stem cells in vitro is provided.
7. The use according to claim 1, characterized in that it is in particular: application of ginsenoside Rg1 and its derivatives in preparing biologic for promoting calcium ion flow of replicative senescent sub-totipotent stem cells in vitro is disclosed.
8. The use according to any one of claims 1 to 7, wherein the purity of ginsenoside Rg1 is not less than 98%.
9. The use of claim 8, wherein the biologic is a drug.
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