Application of serotonin in promoting cell reprogramming efficiency, cell proliferation and anti-aging, cell rejuvenation and reprogramming method

Abstract

Based on the metabolic factors potentially related to reprogramming in oocytes, the present invention lists specific metabolites in oocytes by means of metabolomics and discovers the reductive metabolic characteristics of oocytes. The present invention provides an application of serotonin in promoting cell reprogramming efficiency, cell proliferation and anti-aging, a method for promoting cell rejuvenation and reprogramming, and relates to the field of biotechnology. The present invention proves that including metabolites such as appropriate concentrations of nicotinamide mononucleotide, taurine, serotonin and the oocyte extract itself can promote cell rejuvenation. Among them, especially serotonin, can promote cell reprogramming and cell rejuvenation, extend the lifespan of mice, and reduce the aging level of tissues and organs. Therefore, the present invention finds small molecule metabolites that can promote cell reprogramming and cell rejuvenation through oocytes, which is safer and more reliable than the method independent of gene editing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an application of serotonin in promoting cell reprogramming efficiency, cell proliferation and anti-aging, and a method for promoting cell rejuvenation and reprogramming. Background Art

[0002] Aging is an inevitable process that affects all organisms, manifested as multi-organ degeneration and decline in physiological functions, ultimately leading to an increase in the risk of chronic diseases and death. Therefore, it is very necessary to understand the occurrence and development of aging and how to intervene in aging to extend the life expectancy of humans.

[0003] In recent years, remarkable progress has been made in aging-related research, including the identification of aging phenotypes to studying the genetic background behind these phenotypes, the identification of genes related to biological lifespan, and the intervention in lifespan based on targeting related genes and pathways. For example, the clearance of senescent cells (senolysis) is carried out through the combined treatment of venetoclax, dasatinib and quercetin (D / Q), fisetin, cardiotonic glycosides and other drugs, or the mTOR signaling pathway is inhibited and the insulin / IGF-1 signal is reduced through calorie restriction (CR). Recent research has provided new ideas for anti-aging research based on the fact that reprogramming can restore the epigenetic clock, metabolic homeostasis and transcriptome changes of somatic cells. However, the reprogramming methods used in these studies are all induced reprogramming mediated by the Yamanaka reprogramming factors OSKM (Oct3 / 4, Sox2, Klf4 and c-Myc), and the risk is that complete induced reprogramming will cause somatic cells to lose their original identity characteristics and become pluripotent and differentiate in an undirected manner, thus causing the risk of tumorigenesis. Similar to the induced reprogramming mediated by Yamanaka factors, somatic cell nuclear transfer (SCNT) is another reprogramming method, that is, a process of finally generating totipotent cells by putting the nucleus of terminally differentiated somatic cells into enucleated oocytes. Since the expression level of Yamanaka factors in oocytes is very low, this indicates that there must be other factors promoting reprogramming in oocytes. Combining the more reduced metabolic characteristics of oocytes compared to other stages of early embryonic development and the precise regulation of metabolism on early embryonic development, the factors promoting totipotency shaping in somatic cell nuclear transfer may not only be related to epigenetic remodeling, but may also involve specific metabolites and metabolic pathways. Therefore, using oocyte metabolites may be a new idea for promoting cell rejuvenation and reprogramming.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide the application of 5-hydroxytryptamine (5-HT) in promoting the reprogramming efficiency of human cells.

[0006] The second object of the present invention is to provide a cell reprogramming method.

[0007] The third object of the present invention is to provide a culture medium for cell reprogramming.

[0008] The fourth object of the present invention is to provide a method for promoting cell rejuvenation based on oocyte-specific metabolites;

[0009] The fifth object of the present invention is to provide the application of 5-hydroxytryptamine in promoting the proliferation of human cells.

[0010] The sixth object of the present invention is to provide the application of 5-hydroxytryptamine in the preparation of anti-aging products.

[0011] In order to achieve the above objects, the following technical solutions are specifically adopted:

[0012] In the first aspect, the present invention provides the application of 5-hydroxytryptamine in promoting the reprogramming efficiency of human cells; the human cells include human dermal fibroblasts and human skin fibroblasts.

[0013] As a further technical solution, the reprogramming includes OSKM-induced reprogramming.

[0014] In the second aspect, the present invention provides a cell reprogramming method, in which human cells are cultured with a human cell culture medium containing 5-hydroxytryptamine during the process of OSKM-induced reprogramming of human cells.

[0015] As a further technical solution, the human cells include fibroblasts.

[0016] In the third aspect, the present invention provides a culture medium for cell reprogramming, and the culture medium is a human cell culture medium containing 5-hydroxytryptamine.

[0017] In the fourth aspect, the present invention provides a method for promoting cell rejuvenation based on oocyte-specific metabolites, and human cells are cultured with a human cell culture medium containing oocyte-specific metabolites;

[0018] The oocyte-specific metabolites include 5-hydroxytryptamine.

[0019] As a further technical solution, the human cells include mesenchymal stem cells.

[0020] In the fifth aspect, the present invention provides the application of 5-hydroxytryptamine in promoting the proliferation of human cells; the cells include mesenchymal stem cells.

[0021] In the sixth aspect, the present invention provides the application of 5-hydroxytryptamine in the preparation of anti-aging products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Based on the metabolic factors potentially related to reprogramming in oocytes, the present invention lists specific metabolites in oocytes by means of metabolomics and discovers the reductive metabolic characteristics of oocytes. Using mesenchymal stem cells as an in vitro cell model and G608G premature aging mice as an in vivo model, it is demonstrated that including nicotinamide mononucleotide, taurine, serotonin and other metabolites at appropriate concentrations, as well as the oocyte extract itself, can promote cell regeneration. Among them, especially serotonin, treating cells with a concentration of 5 μM for 72 h can promote cell reprogramming and cell regeneration, and feeding G608 mice at 0.375 mg / kg / day can extend the lifespan of the mice and reduce the aging level of tissues and organs in G608G premature aging mice and naturally aging mice. Therefore, the present invention finds small molecule metabolites that can promote cell reprogramming and cell regeneration through oocytes, which is safer and more reliable than the method independent of gene editing. Brief Description of the Drawings

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 In A, it is the principal component analysis diagram of targeted metabolomics, depicting the clustering of three biological replicates of MII oocytes and two-cell stage embryos (n = 124 metabolites per sample); Figure 1 In B, it is the volcano plot analysis of various metabolites in MII oocytes and two-cell embryos. Blue dots indicate metabolites with higher contents at the two-cell stage, red dots indicate metabolites specifically enriched at the MII oocyte stage, and gray dots indicate metabolites with little difference in abundance between the two stages;

[0026] Figure 2 In A-F, it is the comparison of the relative contents of specific metabolites in MII oocytes and two-cell embryos, and statistical analysis is performed using unpaired t-test, with 3 biological replicates in each group;

[0027] Figure 3 Verify the effects of oocyte extract, nicotinamide mononucleotide, taurine and serotonin on the transcriptional levels of aging-related factors and inflammation-related factors by RT-qPCR;

[0028] Figure 4 Effect of serotonin on the efficiency of OSKM reprogramming; in which A shows the reprogramming induction of human dermal fibroblasts by lentivirus infection. The bar graph represents the number of clones and alkaline phosphatase levels generated after the induction was completed (day 14 of reprogramming). The third picture from the left is the bright-field schematic diagram of the clones in the control group, and the fourth picture from the left is the bright-field schematic diagram of the clones in the serotonin-treated group. Paired t-test was used for statistical analysis, with 3 biological replicates in each group; B shows the reprogramming induction of human skin fibroblasts by lentivirus infection. The bar graph represents the number of clones and alkaline phosphatase levels generated after the induction was completed (day 14 of reprogramming). The third picture from the left is the bright-field schematic diagram of the clones in the control group, and the fourth picture from the left is the bright-field schematic diagram of the clones in the serotonin-treated group. Paired t-test was used for statistical analysis, with 3 biological replicates in each group;

[0029] Figure 5 Verification of the anti-aging effect of serotonin on the pathological model of mesenchymal stem cells from premature aging patients; in which A shows the SA-β-Gal staining (upper) and statistics (lower) of mesenchymal stem cells in each group. One-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; B shows the CCK-8 detection results of mesenchymal stem cells in each group. Two-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; C shows the transcriptional levels of IL1B, IL6, CDKN2A, CDKN1A, and TP53 in mesenchymal stem cells in each group detected by RT-qPCR. One-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group;

[0030] Figure 6 Verification of the anti-aging effect of serotonin on the replicative model of normal human mesenchymal stem cells. In which A shows the SA-β-Gal staining (upper) and statistics (lower) of mesenchymal stem cells in each group. One-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; B shows the CCK-8 detection results of mesenchymal stem cells in each group. Two-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; C shows the transcriptional levels of IL1B, IL6, CDKN2A, CDKN1A, and TP53 in mesenchymal stem cells in each group detected by RT-qPCR. One-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group;

[0031] Figure 7To verify the delay of serotonin in individual-level aging in the in-vivo model of G608G premature aging mice; where A is the survival curve of 23 G608G mice fed with serotonin and 22 G608G mice not fed with serotonin; B is the open field trajectory map of each group of mice; C is the Masson staining map of the lungs, muscles and aorta of each group of mice; D is the quantitative analysis of the fibrosis degree of the lungs, muscles and aorta in C, and one-way ANOVA test is used for statistical analysis, with 3 biological replicates in each group;

[0032] Figure 8 In A, it is the HE staining map of the liver, spleen and aorta of each group of G608G mice; Figure 8 In B, it is the quantitative analysis of the number of central veins per unit area in the liver, the number of vascular smooth muscle cells per unit area in the aorta, and the percentage of the germinal center area of the spleen in A, and one-way ANOVA test is used for statistical analysis, with 5 biological replicates in each group;

[0033] Figure 9 To verify the delay of serotonin in individual-level aging in the in-vivo model of naturally aging mice; where A is the Masson staining map of the lungs, muscles and aorta of each group of mice; B is the quantitative analysis of the fibrosis degree of the lungs, muscles and aorta in A, and one-way ANOVA test is used for statistical analysis, with 3 biological replicates in each group;

[0034] Figure 10 In A, it is the HE staining map of the liver, spleen and aorta of each group of naturally aging mice; Figure 10 In B, it is the quantitative analysis of the number of central veins per unit area in the liver, the number of vascular smooth muscle cells per unit area in the aorta, and the percentage of the germinal center area of the spleen in A, and one-way ANOVA test is used for statistical analysis, with 3 biological replicates in each group;

[0035] Figure 11 In A, it is the protein level of mitochondrial regeneration-related factors detected by Western Blot method in three groups of cells: WT MSCs, G608G MSCs and G608G MSCs + 5-HT; Figure 11 In B, it is the detection result of ATP level in three groups of cells: WT MSCs, G608G MSCs and G608G MSCs + 5-HT; Figure 11 In C, it is the detection result of OCR level in three groups of cells: WT MSCs, G608G MSCs and G608G MSCs + 5-HT; Figure 11 In D, it is the statistical result of the basal respiration value and the maximum respiration value in C;

[0036] Figure 12A in it is the qPCR results of all 5-HTR subtypes; Figure 12 B in it is the transcriptome analysis results of all 5-HTR subtypes; Figure 12 C in it is the CCK8 cell proliferation results of four groups of cells, namely G608G MSCs, G608G MSCs + 5-HT, and G608G MSCs + 5-HT + SB; Figure 12 D in it is the protein expression results related to mitochondrial regeneration function in four groups of cells, namely WT MSCs, G608G MSCs, G608G MSCs + 5-HT, and G608G MSCs + 5-HT + SB;

[0037] Figure 13 A in it is the GO analysis of up-regulated genes in G608G MSCs vs WT MSCs; Figure 13 B in it is the GO analysis of down-regulated genes in G608G MSCs + 5-HT vs G608G MSCs;

[0038] Figure 14 A in it is the flow chart of click chemistry experiment; Figure 14 B in it is the mass spectrometry qualitative analysis (top 20 proteins) of serotoninylated peptide segments enriched by click chemistry method; Figure 14 C in it is to further detect serotoninylated HSP90AB1 by IP / WB;

[0039] Figure 15 Mass spectrometry analysis shows that the Gln (Q) residue is serotoninylated at the 207th amino acid site;

[0040] Figure 16 Mass spectrometry analysis shows that the Gln (Q) residue is serotoninylated at the 493rd amino acid site;

[0041] Figure 17 Taking G608GshHSP90AB1 as the control group and G608GshHSP90AB1 + WT, G608GshHSP90AB1 + Q207A, and G608GshHSP90AB1 + Q493A three kinds of cells as the experimental groups, the transcriptional levels of endoplasmic reticulum stress-related genes including DDIT3, ATF4, and PPP1R15A are detected under the conditions of 5-HT treatment or not;

[0042] Figure 18Using G608GshHSP90AB1 as the control group, and G608GshHSP90AB1+WT, G608GshHSP90AB1+Q207A, G608GshHSP90AB1+Q493A as the experimental groups, the ratio of β-galactosidase positive cells in each group was detected under the conditions of 5-HT treatment or no treatment. Among them, A is the bright field result diagram of β-gal; Figure 18 B in Figure 18 is the statistical chart of A in Specific implementation manners

[0043] The embodiments of the present invention will be described in detail below in combination with the implementation manners and examples. However, those skilled in the art will understand that the following implementation manners and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0044] Term explanation:

[0045] Oocyte: The oogonium that undergoes meiosis during oogenesis. Oocytes are divided into primary oocytes, secondary oocytes and mature oocytes. MII: Mature oocytes arrested at the metaphase of the second meiosis. Zygote: The fertilized egg at the cell stage. Two-cell: After embryo fertilization, the initial division will produce two cells, forming a two-cell embryo.

[0046] Reprogramming: It refers to the process of making differentiated cells redifferentiate back to the stem cell state through methods of changing epigenetic modifications without changing the gene sequence, so as to possess pluripotency or even totipotency. Currently, reprogramming mainly includes two types. One is the induced reprogramming mediated by the Yamanaka reprogramming factors OSKM (Oct3 / 4, Sox2, Klf4 and c-Myc); the other is the somatic cell nuclear transfer (SCNT) technology.

[0047] Rejuvenation: It refers to the process of making cells or collective tissues and organs regain vitality through some intervention means such as reprogramming, small molecules, etc.

[0048] In the first aspect, the present invention provides the application of 5-hydroxytryptamine in promoting the reprogramming efficiency of human cells; the human cells include human dermal fibroblasts and human skin fibroblasts.

[0049] The inventors' research found that during the process of human cell reprogramming, adding extra serotonin to the culture medium can significantly improve the reprogramming efficiency.

[0050] In some alternative embodiments, the reprogramming includes OSKM-induced reprogramming.

[0051] In some alternative embodiments, the human cells include but are not limited to fibroblasts, or other cells well-known to those skilled in the art that can be reprogrammed by OSKM induction.

[0052] In a second aspect, the present invention provides a method for cell reprogramming. During the process of OSKM-induced cell reprogramming, human cells are cultured in a human cell culture medium containing serotonin.

[0053] During the process of OSKM-induced cell reprogramming, culturing fibroblasts in a human cell culture medium containing serotonin can effectively improve the reprogramming efficiency of the cells.

[0054] In some alternative embodiments, the human cells include but are not limited to fibroblasts, or other cells well-known to those skilled in the art that can be reprogrammed by OSKM induction.

[0055] The present invention does not specifically limit the concentration of serotonin in the culture medium, and an appropriate concentration can be selected according to the cells to be reprogrammed. In some alternative embodiments, the concentration of serotonin in the culture medium can be, for example, 5 μM.

[0056] In some alternative embodiments, the human cell culture medium is mTeSR™1 Basal Medium (STEMCELL, 85851) containing 5 μM serotonin.

[0057] In a third aspect, the present invention provides a culture medium for cell reprogramming, and the culture medium is a human cell culture medium containing serotonin.

[0058] This culture medium is used for cell reprogramming and can effectively improve the reprogramming efficiency of fibroblasts and the like.

[0059] In some alternative embodiments, the human cell culture medium includes mTeSR™1 Basal Medium (STEMCELL, 85851) containing 5 μM serotonin.

[0060] The present invention does not specifically limit the concentration of serotonin in the culture medium, and an appropriate concentration can be selected according to the cells to be reprogrammed. In some alternative embodiments, the concentration of serotonin in the cell culture medium can be, for example, 5 μM.

[0061] Fourth aspect, the present invention provides a method for promoting cell rejuvenation based on oocyte-specific metabolites, which comprises culturing human cells in a human cell culture medium containing oocyte-specific metabolites;

[0062] The oocyte-specific metabolites include serotonin.

[0063] Through the research of the inventors, it is found that nicotinamide mononucleotide, taurine and serotonin can reduce the expression of senescence and inflammatory factors to a certain extent and have the effect of promoting cell rejuvenation.

[0064] In some alternative embodiments, the human cells include mesenchymal stem cells.

[0065] In some alternative embodiments, the culture medium is a low-glucose DMEM medium (Gibco, C11885500BT) containing the oocyte-specific metabolites, 10% fetal bovine serum (FBS), and 100x Gluta-MAX (Gibco, 25030081).

[0066] The present invention does not specifically limit the concentration of serotonin in the culture medium, and an appropriate concentration can be selected according to the cells to be reprogrammed. In some alternative embodiments, the concentration of serotonin in the culture medium can be, for example, 5 μM.

[0067] Fifth aspect, the present invention provides the application of serotonin in promoting cell proliferation; the cells include mesenchymal stem cells.

[0068] Through the research of the inventors, it is found that during the culture process, treating cells with serotonin can significantly increase the cell proliferation rate.

[0069] Sixth aspect, the present invention provides the application of serotonin in the preparation of anti-aging products.

[0070] Through the research of the inventors, it is found that serotonin can reduce the expression of senescence-related factors and alleviate the senescence phenotype of tissues, and can be used to prepare anti-aging drugs.

[0071] The present invention will be further illustrated by specific examples below.

[0072] It should be noted that in the following examples, "sh" represents knockdown.

[0073] Examples

[0074] I. Collect MII oocytes and embryos at two different stages of 2-cell for metabolomics analysis

[0075] Four-week-old (4W) C57BL / 6 female mice were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 5 IU). After 48 h, human chorionic gonadotropin (hCG, 5 IU) was injected. Metaphase II (MII) oocytes and two-cell embryos were collected at 14 h and 40 h after the injection of human chorionic gonadotropin, respectively. The collection methods were as follows:

[0076] MII oocytes: The mice were humanely sacrificed by cervical dislocation, and the bilateral oviducts were removed. Under a stereomicroscope, the swollen ampulla of the oviduct was torn open with forceps to release the oocytes with cumulus cells. The oocytes were placed in a 0.3 mg / ml hyaluronidase solution for about 1 minute, transferred to fresh M2 medium (sigma, catalog number M7167), and pipetted several times until the cumulus cells detached. The oocytes with polar bodies were collected with a pipette and washed 2-3 times in fresh M2 medium to remove excess impurities, then washed three times in 0.9% NaCl to remove excess proteins, and then transferred to a low-attachment 1.5 ml EP tube. An appropriate amount of pre-cooled pure methanol was added and stored at -80 °C. 100 oocytes were collected for each group, with three replicates.

[0077] Two-cell embryos: At 40 h after the injection of human chorionic gonadotropin, two-cell embryos were collected by flushing the oviducts with a needle. Similarly, three replicates were collected, with 100 embryos for each group.

[0078] The collected MII oocytes or two-cell embryos were centrifuged at 15,000 rpm for 15 minutes in a 4°C low-temperature centrifuge. Gently transfer the supernatant to a new centrifuge tube, dry it with a vacuum pump at 30°C, and add 80% methanol. Then evaporate the methanol to dryness, and resuspend the dried metabolites in 30 μl of 0.03% deionized water and shake. Then centrifuge at 15,000 rpm for 15 minutes in a 4°C low-temperature centrifuge, and take the supernatant for liquid chromatography-mass spectrometry (Liquid Chromatography Mass Spectrometry, LC–MS / MS) detection. The liquid chromatography experiment was performed on an ultra-high resolution liquid chromatography system, where the chromatographic column was an ACQUITY UPLC HSS-T3 UPLC column (150×2.1 mm, 1.8 μm, Waters), and the mobile phase gradient was successively 1% mobile phase B from 0–3 min; 1–99% mobile phase B from 3–15 min; 99% mobile phase B from 15–17 min; 99–1% mobile phase B from 17–17.1 min; 1% mobile phase B from 17.1–20 min. Mobile phase A was 0.03% formic acid / water solution, and mobile phase B was 0.03% formic acid / acetonitrile solution. The flow rate was 0.25 ml / min, the chromatographic column was maintained at 35°C, the samples in the autosampler were maintained at 4°C, and the injection volume was 20 μL. Mass spectrometry detection was performed using a SCIEX QTRAP 6500+ mass spectrometer, and the multiple reaction monitoring mode (MRM) of mass spectrometry was used. The chromatogram was examined and the peak areas were calculated using MultiQuant software v.3.0 (SCIEX). The peak area of each detected metabolite was normalized to the total ion count of the sample. Therefore, the obtained content was the ratio between the detected metabolite and the total metabolite.

[0079] Through metabolomics analysis, relevant data on 124 metabolites were obtained in total. Principal component analysis showed that the embryonic metabolites in the two stages could be well distinguished ( Figure 1 in A), and compared with two-cell embryos, MII oocytes were enriched with more metabolites related to reduction, including nicotinamide adenine dinucleotide (NAD+ / NADH), vitamin C (Ascorbic acid), taurine, polyamines (spermine, spermidine), serotonin (Serotonin, 5-HT), etc. ( Figure 1 in B, Figure 2in A-F). Metabolite enrichment analysis also showed that the nicotinic acid and nicotinamide pathway, glutathione pathway, taurine pathway, arginine pathway, and tryptophan pathway were more active during the oocyte stage. Overall, compared with the two-cell stage after fertilization, MII oocytes tended to be enriched in reducing metabolites and had antioxidant properties. This antioxidant property is a common feature of many metabolites that promote reprogramming and anti-aging, such as vitamin C and taurine.

[0080] II. Promoting cell rejuvenation using oocyte extracts and oocyte-specific metabolites

[0081] Using mesenchymal stem cells differentiated from iPSCs (induced pluripotent stem cells) derived from normal individuals (WT) and premature aging patients (G608G, LMNA c. G608G C>T) as an in vitro cell model for research, mesenchymal stem cells derived from normal individuals (WT MSCs) were used as a control, and mesenchymal stem cells derived from premature aging patients (G608G MSCs) were used as the experimental group to explore the role of metabolites in pathological aging, mainly detecting the expression level of β-galactosidase, cell proliferation rate, and the expression of factors related to the senescence-associated secretory phenotype (SASP).

[0082] G608G MSCs were respectively seeded in a medium (low-glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS, Fetal Bovine Serum), 100x Gluta-MAX (Gibco, 25030081)) containing oocyte extract, or 5 μM serotonin, or 200 μM nicotinamide mononucleotide or 1.25 mM taurine and cultured for 72 h, and a control group was set up. Then, qPCR was used to detect senescence-related factors CDKN2A (P16), CDKN1A (P21), TP53 (P53) and inflammation-related factors IL6 , IL1B . The transcriptional levels were as shown in Figure 3 . It was found that these metabolites specifically enriched in oocytes could reduce the expression of senescence and inflammation factors to a certain extent. Similarly, oocyte extract also had a certain effect, but the effect was not as obvious as that of a single metabolite, which might be because there were also many other unrelated metabolites in the oocyte extract.

[0083] Note: The oocyte extract was obtained by lysing 100 oocytes with 80% pre-cooled methanol, centrifuging to take the supernatant, drying it, and then re-dissolving it with the medium.

[0084] III. Oocyte-specific metabolite serotonin promotes reprogramming efficiency

[0085] The oocyte-specific metabolite serotonin has the effect of promoting reprogramming and cell rejuvenation.

[0086] After obtaining the corresponding virus by transfecting 293T cells with the lentiviral plasmid of Yamanaka factors OSKM, human skin fibroblasts (NHDF) from young people and human dermal fibroblasts (AHDF) from the elderly were induced to reprogram. On the first day of the start of reprogramming, additional serotonin (5 μM, continuously added throughout the reprogramming process) was added to the mTeSR™1 Basal Medium (STEMCELL, 85851) to explore the effect of this metabolite on the reprogramming efficiency. During the experiment, the culture without serotonin addition was used as a control (Ctr). Around the 14th day of reprogramming (when clones could be seen under bright field), the number of reprogrammed clones ( Figure 4 A in Figure 4 and Figure 4 A in Figure 4 B), the level of alkaline phosphatase (AP) (

[0087] B in

[0088] IV. The rejuvenating effect of the oocyte-specific metabolite serotonin

[0089] Using mesenchymal stem cells differentiated from iPSCs derived from normal individuals (WT) and patients with premature aging (G608G, LMNA c. G608G C>T) as an in vitro cell model for research, the role of serotonin in pathological aging was explored using G608G MSCs as the experimental group.

[0090] The experimental steps include: inoculating G608G MSCs in a low-glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS, Fetal Bovine Serum) and 100x Gluta-MAX (Gibco, 25030081) for culture, then treating G608G MSCs with serotonin (final concentration of 5 μM) for 72 h, and using WT MSCs and G608G MSCs without serotonin treatment as controls. Subsequently, the effects on a series of cell senescence-related markers such as the expression level of β-galactosidase, cell proliferation rate, and the expression levels of factors related to the senescence-associated secretory phenotype (SASP) were detected.

[0091] The experiments involved mainly include:

[0092] β - cell Galactosidase Staining: SA - β - Gal staining was performed using an SA - β - Gal staining kit (Beyotime, RG0039, China) according to the manufacturer's protocol. Briefly, 50,000 cells / well were cultured in a 12 - well plate. After washing once with phosphate - buffered saline (PBS), the fixative was added and incubated at room temperature for 15 min. Then, the cells were stained with SA - β - Gal staining solution at pH 6.0 and 37 °C overnight. Senescent cells were observed under a light microscope.

[0093] CCK8 Cell Proliferation: 1000 mesenchymal stem cells per well were seeded onto a 96 - well transparent culture dish. After the cells adhered, the CCK8 solution was added to the medium (1:100), and the cells were incubated at 37 °C for 4 h. The absorbance was measured at 450 nm. The second measurement was taken after 24 h, and a total of four measurements were taken. Then, the cell number was calculated according to the standard curve.

[0094] RT - qPCR: Total cellular RNA was extracted using the phenol - chloroform method or a commercial kit, and then reverse - transcribed to obtain cDNA. The expression of target genes was detected using TB Green Premix (RR820A, TAKARA) on a LightCycler 480 Instrument II System (Roche) or a QuantStudio 6 Flex machine (ABI). All genes were normalized using ACTIN as an internal reference gene.

[0095] Figure 5 β - galactosidase staining experiments ( Figure 5 in A), CCK8 cell proliferation experiments ( Figure 5 in B), and qPCR ( Figure 5 in C) experiments showed that serotonin could reduce the number of β - gal - positive cells in pathological senescence, promote cell proliferation, and decrease the expression of senescence - related factors.

[0096] Mesenchymal stem cells differentiated from iPSCs derived from normal humans (WT) were passaged. Young - passage WT MSCs at passages P3 - P5 (denoted as WT Y) and old - passage WT MSCs at passages P12 - P15 (denoted as WTO) were obtained respectively. It should be noted that P here refers to the passage number. All MSCs are called P0 after being induced by iPSC differentiation, and one is added for each subsequent passage.

[0097] Subsequently, WT MSCs of P12 - P15 old passages were inoculated into low - glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS, Fetal Bovine Serum) and 100x Gluta - MAX (Gibco, 25030081) for culture. Then, they were treated with serotonin (final concentration of 5 μM) for 72 h, and WT Y and WT O without serotonin treatment were used as controls. Subsequently, the effects of a series of cellular senescence - related markers such as β - galactosidase expression level, cell proliferation rate, and expression levels of factors related to the senescence - associated secretory phenotype (SASP) were detected.

[0098] Figure 6 Using β - galactosidase ( Figure 6 A in Figure 6 ), CCK8 cell proliferation assay ( Figure 6 B in

[0099] ), and qPCR to detect senescence - related factors ( Figure 7 C in Figure 7 ), it was demonstrated that serotonin also plays a role in replicative senescence.

[0100] The experiments involved mainly included:

[0101] Tissue staining: Tissue samples were fixed in 4% PFA overnight and dehydrated in 70% ethanol for at least 24 h. Then the samples were embedded in paraffin, sectioned, and subjected to HE or Masson staining.

[0102] Open field test: In a white plexiglass box with a length, width, and height of 40 cm each, the open field test was used to evaluate the locomotor activity of mice. The mice were placed in the center of the box floor and allowed 10 minutes to freely explore, and their every move was recorded by a camera. The total distance traveled and the movement trajectory of the mice were analyzed using AlsVision software.

[0103] The results of in vivo experiments showed that long-term drinking of water containing serotonin by premature aging mice (LMNA G608G) could increase their maximum lifespan ( Figure 7 A in [reference], analyzed by Log-rank (Mantel-Cox) test). The open field test also showed that serotonin enhanced the locomotor ability of premature aging mice ( Figure 7 B in [reference]), and the results of Masson staining showed that serotonin could reduce the degree of fibrosis in the lungs, muscles, and aorta at the tissue level ( Figure 7 C-D in [reference]), and the results of HE staining further showed that long-term oral administration of serotonin could increase the blood flow in the liver of premature aging mice, the number of vascular smooth muscle cells per unit area of the aorta, and the germinal center of the spleen ( Figure 8 A-B in [reference]).

[0104] In addition, we fed 12-month-old wild-type C57BL / 6 mice with water containing serotonin (0.375 mg / kg / day) until the mice were 20 months old. Mice not fed with serotonin were used as controls, and anatomical experiments were conducted. It was found that compared with 4-week-old (denoted as 4W) C57BL / 6 mice, 20-month-old mice not fed with serotonin (denoted as 20M) showed obvious histological aging characteristics, while 20-month-old mice fed with serotonin (denoted as 20M+5-HT) could well alleviate the tissue aging phenotype. The results of Masson staining showed that serotonin reduced the degree of fibrosis in the lungs, muscles, and aorta at the tissue level ( Figure 9 A-B in [reference]), and the results of HE staining further showed that long-term oral administration of serotonin could increase the blood flow in the liver of mice, the number of vascular smooth muscle cells per unit area of the aorta, and the germinal center of the spleen ( Figure 10 A-B in [reference]).

[0105] 5. Serotonin delays aging through classical and non-classical pathways

[0106] Classical pathway: 5-HT regulates mitochondrial function depending on 5HTR1B

[0107] After the phenotypic verification of the rejuvenation effect of 5-HT in in vivo and in vitro models, in order to further explore the mechanism of action of 5-HT, transcriptomic analysis was performed on three groups of cells: WT MSCs, and G608G MSCs treated or untreated with 5-HT. The results showed that 5-HT could increase the expression levels of factors related to mitochondrial function. To further prove the relationship between 5-HT and mitochondria, Western Blot was used to detect transcription factors related to mitochondrial regeneration, including PGC1α (peroxisome proliferator-activated receptor γ coactivator 1α), Tomm20 (mitochondrial outer membrane translocase 20), and Timm23 (mitochondrial inner membrane translocase 23) ( Figure 11 A in Figure 11 ), and it was found that 5-HT could increase the protein expression levels of these genes. At the same time, ATP level detection also showed that 5-HT could increase the ATP content in senescent cells ( Figure 11 B in Figure 11 ). To further confirm the effect of 5-HT on mitochondrial function, the OCR (oxygen consumption rate) levels of three groups of cells, WT MSCs, G608G MSCs, and G608G MSCs + 5-HT, were detected by seahorse experiment (

[0108] C in Figure 12 ). The results showed that compared with WT MSCs, the basal respiration level and maximum respiration level of G608G MSCs were both decreased, while 5-HT could indeed enhance mitochondrial respiratory function to a certain extent, including increasing the basal respiration value and maximum respiration value ( Figure 11 D in Figure 12

[0108] ). It is a common way of action of 5-HT that extracellular serotonin activates intracellular signaling pathways by interacting with various subtypes of receptors and then participates in biological processes. To further understand which receptor 5-HT uses to relieve cell senescence, the transcript levels of all 5-HTR (5-hydroxytryptamine receptor) subtypes were analyzed using the transcriptomic data above and verified by qPCR. The results showed that the receptor subtype with the highest expression level in MSCs was 5HTR1B, and the expression levels of other subtypes were very low or almost non-existent ( Figure 12 A in Figure 12 B in ). Therefore, the competitive inhibitor of 5HTR1B, SB-224289 hydrochloride (MCE, HY-101105A, hereinafter abbreviated as SB), was selected to explore whether 5HTR1B mediated the promotion of cell rejuvenation by 5-HT. Referring to the experimental method in the fourth part above, 2 μM of SB was added while treating G608G MSCs with 5-HT, and it was found that the promoting effect of 5-HT on cell proliferation was partially inhibited by SB ( Figure 12where C is the result of CCK8 cell proliferation), including the upregulation of protein expression related to mitochondrial regeneration function was also inhibited ( Figure 12 as shown in D).

[0109] The main experiments involved are as follows:

[0110] ATP assay: MSCs were cultured in 12-well plates for 72 hours. According to the instructions of the ATP detection kit (S0026, Beyotime), whole cells were lysed using lysis buffer. After centrifugation, the supernatant was taken and added to the ATP detection solution, and the luminescence value was read using a Spark multimode microplate reader. Protein content was determined according to the Pierce BCA Protein Assay Kit.

[0111] Cell bioenergetics analysis: Real-time mitochondrial respiration analysis was performed using a Seahorse XF96 Extracellular Flux Analyzer (Agilent, SantaClara, USA) according to the manufacturer's protocol. Briefly, the oxygen consumption rate (OCR) per minute in cell cultures was measured by sequentially adding the following respiratory regulators to determine bioenergetics parameters: 1.5 µM oligomycin (ATP synthase inhibitor; catalog number 103015-100, Agilent), 1 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, an ionophore and mitochondrial uncoupler), and 0.5 µM rotenone (mitochondrial complex I inhibitor). MSCs were seeded at a density of 1.2×10^4 cells / well in 150 µL of the corresponding medium. Before the experiment, the medium was replaced with DMEM without sodium bicarbonate and supplemented with 1 mM pyruvate and 5 mM glucose. Mitochondrial respiration parameters (basal respiration capacity, maximal respiration capacity) were calculated using the OCR values.

[0112] Non-classical pathway: 5-HT inhibits endoplasmic reticulum stress through serotonylation of HSP90AB1, thereby delaying aging.

[0113] Further analysis of the above RNA-seq found that compared with young cells, genes related to "unfolded protein response" and "endoplasmic reticulum stress" were significantly enriched in G608G MSCs, and 5-HT rescued this phenomenon ( Figure 13 where A is the GO analysis of upregulated genes in G608G MSCs vs WT MSCs, Figure 13In B (GO analysis of down-regulated genes in G608G MSCs + 5-HT vs G608G MSCs). It is known that 5-HT can modify proteins through covalent transamination mediated by transglutaminase (TGM2). On the basis of establishing the click chemistry method, G608G MSCs or WT MSCs were treated with 5-PT (5-HT with an alkyne group, MCE, HY-156562A) to label the target protein. After that, the target protein was obtained by immunoprecipitation and then identified by liquid chromatography-tandem mass spectrometry (chromatography-LC-MS / MS) for serotonylated proteins ( Figure 14 In A is the flow chart of the click chemistry experiment). The results showed that the proteins with the highest enrichment in the target proteins included not only ACTIN, which has been previously confirmed to be serotonylated, but also HSP90AB1 (heat shock protein 90 alpha family class B member 1), which is highly related to endoplasmic reticulum stress and the unfolded protein response (B in Figure 14). IP and WB further confirmed that 5-HT can serotonylate HSP90AB1 in WT MSCs and G608G MSCs (C in Figure 14, where the upper part shows the binding of 5-HT and HSP90AB1 in G608G MSCs, and the lower part shows the binding of 5-HT and HSP90AB1 in WT MSCs).

[0114] To further determine the serotonylation site on HSP90AB1, an in vitro serotonylation experiment was carried out using LC-MS / MS. Specifically, the in vitro purified HSP90AB1 protein was co-incubated with 5-HT and recombinant TGM2 enzyme at 37 °C for modification, and then mass spectrometry analysis was performed. The results showed that the Gln (Q) residue was at the 207th ( Figure 15 ) and the 493rd amino acid ( Figure 16) is serotonylated at the site. To prove which site of 5-hydroxytryptamination contributes to the role of 5-HT in promoting cell regeneration, wild-type HSP90β with a Flag tag and HSP90β with the 207th or 493rd glutamine mutated to alanine were further constructed, denoted as WT, Q207A, and Q493A mutants respectively. Wild-type (WT) and Q207A and Q493A mutant HSP90AB1 with Flag were overexpressed respectively in G608G MSCs (G608GshHSP90AB1) with endogenous HSP90AB1 knocked down, denoted as G608GshHSP90AB1+WT, G608GshHSP90AB1+Q207A, and G608GshHSP90AB1+Q493A. Then, these three groups of cells were treated with 5-HT respectively, and G608G MSCs with HSP90AB1 knocked down were used as the control group. It was found that only after the 207th glutamine was mutated to alanine, the effect of 5-HT serotonylating HSP90AB1 was completely inhibited. And the mutant Q207A replacing endogenous wild-type HSP90AB1 indeed eliminated the effect of 5-HT promoting regeneration, manifested as the loss of the regulatory effect on endoplasmic reticulum stress and unfolded protein response genes, including that DDIT3 (DNA damage-inducible transcript factor 3), ATF4 (transcriptional activator 4), and PPP1R15A (protein phosphatase 1 regulatory subunit 15A) were no longer downregulated. However, the Q493A mutant did not have this effect ( Figure 17 ). According to the same experimental design, the effect of mutating the 207th glutamine to alanine on the role of 5-HT was further detected. The results showed that Q207A inhibited the effect of 5-HT in reducing the proportion of β-Gal positive cells ( Figure 18 In A is the bright-field result graph of β-gal, and in B is the statistical graph).

[0115] The experiments involved mainly include:

[0116] 5-PT Immunoprecipitation Experiment: Serotonylated proteins were labeled using copper-catalyzed click chemistry. The alkyne-functionalized 5-HT derivative 5-PT was conjugated with biotin-azide molecules, which was prepared by MedChemExpress (MCE®). Cells were treated with 10 µM 5-PT for 72 hours. Cell pellets were sonicated in 100 µL click reaction buffer (Thermo, C10643), then centrifuged at 15,000 rpm for 15 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube. 0.25 mM biotin-azide, 0.1 mM CuSO4, and 50 μL of 1× click additive were sequentially added to the lysate, and the final volume was adjusted to approximately 500 μL with click reaction buffer. The samples were incubated at 4°C for 2 - 3 hours. Magnetic streptavidin beads (ThermoFisher, 11206D) were washed twice in PBS. The reaction volume was adjusted to 1 mL with PBS, and 5% was taken as the input control before adding the washed beads. 20 μL of magnetic bead suspension was used for each immunoprecipitation, and the samples were incubated with rotation at room temperature for 3 hours. After incubation, the samples were placed on a magnetic rack to separate the beads from the supernatant, and the supernatant was carefully removed. The beads were washed three times with cold RIPA buffer containing protease inhibitors (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM PMSF), and then washed three times with cold DPBS to remove excess detergent. After the last wash, 2× SDS was added to the beads and boiled at 98°C for 8 minutes, followed by gel electrophoresis and incubation with appropriate primary and secondary antibodies.

[0117] In Vitro Enzymatic Serotonylation Experiment: 5-HT (5 mmol / L), 0.25 mg of functional TGM2 protein (MCE, HY-P76114), and HSP90AB1 protein (abcam, ab80033) were co-incubated at 37°C to transamidate 5-HT to HSP90AB1. The total reaction volume was 25 µL of enzyme reaction buffer (25 mM Tris-HCl, pH 8 and 5 mM CaCl 2 , containing protease inhibitors). The reaction buffer was incubated in the dark at 37°C for 3 hours. The samples were boiled with 2× SDS at 95°C for 10 minutes. Serotonylated proteins were visualized by Coomassie Brilliant Blue staining, followed by LC-MS / MS analysis to detect serotonylation modification.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them.

Claims

1. Application of 5-hydroxytryptamine in promoting the efficiency of human cell reprogramming in vitro; the human cells are human skin fibroblasts; The reprogramming is OSKM-induced reprogramming.

2. A cell reprogramming method, characterized in that: During OSKM-induced human skin fibroblast reprogramming, human skin fibroblasts were cultured in human cell culture medium containing 5-HT.

3. A method for promoting cell regeneration based on oocyte-specific metabolites, characterized in that: Human mesenchymal stem cells were cultured in human cell culture medium containing oocyte-specific metabolites; The oocyte-specific metabolite is 5-hydroxytryptamine.

Images