CN115634221A - Application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation - Google Patents
Application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, relates to application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation, and particularly relates to application of betaine and mesenchymal stem cell in preparing a medicine for treating diseases caused by osteoporosis and obesity. Experimental research proves that the mesenchymal stem cells treated by betaine can increase the number of ALP positive cells and plaque calcified extracellular matrix in the osteogenic differentiation process of the mesenchymal stem cells cultured in vitro, and simultaneously up-regulates the expression levels of OPN, RUNX-2 and OCN proteins in the osteogenic differentiation process of the mesenchymal stem cells. Lipid droplet number and size were reduced while down-regulating the expression of lipid major genes PPAR γ, CEBP α, FASN. The betaine can promote the osteogenesis of the mesenchymal stem cells and inhibit the adipogenic differentiation of the mesenchymal stem cells through a PI3K-Akt signal channel, the interaction of a cytokine-cytokine receptor and the interaction of an ECM receptor, and finally can play a role in relieving diseases caused by osteoporosis and obesity.
Description
Technical Field
The invention belongs to the technical field of biomedicine, relates to application of betaine in regulation of mesenchymal stem cell adipogenesis and osteogenic differentiation, and particularly relates to application of betaine and mesenchymal stem cells in preparation of a medicine for treating diseases caused by osteoporosis and obesity.
Background
Osteoporosis is a systemic and systemic metabolic disease caused by low bone mass content and the increase of fragility of the organism due to the destruction of bone tissue microstructure, and is mainly caused by that the bone formation in the organism skeletal metabolism is smaller than bone resorption, wherein osteoblasts and osteoclasts are main cells for regulating and controlling the processes, the osteoclasts mainly mediate bone resorption to accelerate the occurrence of osteoporosis, and the osteoblasts mainly secrete bone matrix and calcium salt in the process of bone formation to form new mature bones so as to relieve the osteoporosis. Obesity can lead to diabetes, coronary heart disease, and osteoarthritis, thereby increasing the risk of death. As the population ages, the incidence of bone loss and obesity is increasing. However, there is currently no better treatment to increase endogenous bone formation and reduce adipogenesis.
Mesenchymal Stem Cells (MSCs) are a type of pluripotent cells, which can be obtained from adipose tissue, peripheral blood, bone marrow, placenta, umbilical cord, etc., and have the ability to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic cells. Adipose-derived mesenchymal stem cells are widely used because they are easy to separate and have abundant tissue sources. Human adipose mesenchymal stem cells (hascs) are a class of pluripotent stem cells with self-renewal and multipotent differentiation potential. It has been found that human adipose-derived mesenchymal stem cells can be used for the treatment of various diseases, and their therapeutic effects are generally attributed to their cytokine secretion and immunoregulatory effects, rather than their ability to differentiate directionally into target cells. The supernatant of the human adipose-derived mesenchymal stem cell conditioned medium contains various growth factors, cytokines, chemokines, angiogenesis factors, exosomes and the like, and the substances have obvious effects on promoting proliferation, migration and differentiation of cells. In addition, the supernatant does not contain cells, so that the problem of immunological rejection does not exist, and the use is relatively safe.
A number of small molecules have been used to explore the effects on MSCs differentiation. Betaine is an alkaloid and has a chemical name ofN,N,NTrimethyl glycine with chemical structure similar to amino acid and belonging to quaternary ammonium base material and molecular formula of C 5 H 11 NO 2 It is widely present in animals and plants. In the plants, the lycium barbarum and the leguminous plants contain betaine. Beet molasses is the main source of betaine. In animals, liver, spleen and amniotic fluid of mollusks such as octopus, cuttlefish and shrimp, and vertebrates (including human) contain betaine. Betaine (N, N-trimethylglycine) is a natural zwitterionic molecule that is widely distributed in many organisms. Betaine is a methyl donor and permeant, involved in the methionine cycle by betaine-homocysteine methyltransferase (BHMT). Studies by Anthony J. Barak et al indicate that betaine can remethylate homocysteine and remove SAH, potentially for the treatment of liver disease. Betaine treatment prevents the reduction of osteogenic differentiation caused by alcohol-induced femoral head necrosis (ONFH). It has been shown by studies that betaine can prevent the increase of liver lipid accumulation, improve sugar tolerance and insulin sensitivity. However, the molecular mechanisms and effects of betaine on adipogenic and osteogenic differentiation of MSCs are not clear. Therefore, the deep research on the regulation of mesenchymal stem cell adipogenic and osteogenic differentiation by betaine has important significance for treating diseases caused by osteoporosis and obesity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the application of betaine in preventing and treating diseases caused by osteoporosis and obesity by regulating the adipogenic and osteogenic differentiation of Mesenchymal Stem Cells (MSCs). According to the invention, through research, the betaine increases the number of ALP positive cells and plaque calcified extracellular matrix in the osteogenic differentiation process of mesenchymal stem cells cultured in vitro, and simultaneously, the expression levels of OPN, RUNX-2 and OCN proteins in the osteogenic differentiation process of the mesenchymal stem cells are up-regulated. Lipid droplet number and size were reduced while down-regulating the expression of lipid major genes PPAR γ, CEBP α, FASN. The betaine can promote the osteogenesis of the mesenchymal stem cells and inhibit the adipogenic differentiation of the mesenchymal stem cells through a PI3K-Akt signal channel, the interaction of a cytokine-cytokine receptor and the interaction of an ECM receptor, and finally can play a role in relieving diseases caused by osteoporosis and obesity.
In order to achieve the purpose, the invention adopts the following technical scheme.
An accelerant for promoting osteogenic differentiation of mesenchymal stem cells, wherein the accelerant is betaine.
The promoter is used for promoting the differentiation of mesenchymal stem cells into osteoblasts.
Use of betaine in preparing osteogenic differentiation promoter for mesenchymal stem cells is provided.
The application comprises the application of betaine in preparing OPN, RUNX-2 and OCN expression promoters and PPAR gamma, CEBP alpha and FASN expression inhibitors for promoting the osteogenic differentiation of mesenchymal stem cells.
The application of betaine in preparing medicine for preventing and treating osteoporosis and/or obesity.
The application of betaine in preparing medicines for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells.
The betaine and mesenchymal stem cells are applied to the preparation of medicines for treating diseases caused by osteoporosis and/or obesity.
A method for promoting osteogenic differentiation and/or inhibiting adipogenic differentiation of mesenchymal stem cells comprises treating mesenchymal stem cells with betaine.
Further, the betaine is used in the above applications and methods at a concentration of 10mM.
Further, the application and the method for promoting osteogenic differentiation of the mesenchymal stem cells increase the number of ALP positive cells and plaque calcified extracellular matrix in the osteogenic differentiation process of the mesenchymal stem cells, up-regulate the expression levels of OPN, RUNX-2 and OCN proteins in the osteogenic differentiation process of the mesenchymal stem cells, and inhibit the adipogenic differentiation of the mesenchymal stem cells to down-regulate the expression levels of PPAR gamma, CEBP alpha and FASN genes in the adipogenic differentiation process of the mesenchymal stem cells.
Further, in the application and the method, the mesenchymal stem cells are human adipose-derived mesenchymal stem cells hAD-MSCs or human umbilical cord mesenchymal stem cells hUC-MSCs.
A medicine for preventing and treating diseases caused by osteoporosis and/or obesity contains betaine.
A medicine for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells contains betaine.
The medicament for preventing and treating diseases caused by osteoporosis and/or obesity, the medicament for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells and other effective components which are compatible with betaine and play a synergistic role.
The medicine for preventing and treating diseases caused by osteoporosis and/or obesity, the medicine for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells and a pharmaceutically acceptable carrier and/or excipient.
The medicament for preventing and treating the diseases caused by osteoporosis and/or obesity or the medicament for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells is characterized in that the dosage form of the medicament comprises but is not limited to tablets, granules, capsules, dropping pills, sustained release agents, oral liquid preparations and injections.
The mesenchymal stem cells of the present invention are preferably human adipose-derived mesenchymal stem cells hAD-MSCs or human umbilical cord mesenchymal stem cells hUC-MSCs, such as umbilical cord, bone marrow, fat, umbilical cord blood, amnion, placenta, dental pulp, and peripheral blood.
The beneficial effects of the invention are as follows.
The invention provides a new application of betaine, namely, the betaine with different concentrations is used for preventing and treating osteoporosis by promoting osteogenic differentiation of mesenchymal stem cells and/or preventing and treating diseases caused by obesity by inhibiting adipogenic differentiation of adipose mesenchymal stem cells. 10mM betaine significantly increased the amount of ALP positive cells and plaque calcified extracellular matrix with upregulation of OPN, RUNX-2 and OCN. The lipid drop number and size are reduced, and the expression of lipid-forming major genes such as PPAR gamma, CEBP alpha, FASN and the like is simultaneously reduced. The Gene Ontology (GO) analysis shows that in MSCs treated by betaine, adipocyte differentiation and bone mineralization functional items are enriched, KEGG shows that a PI3K-Akt signal channel, a cytokine-cytokine receptor interaction and an ECM-receptor interaction channel are enriched, and the betaine is used as an auxiliary agent for mesenchymal stem cell treatment, can finally play a role in relieving osteoporosis, can be used for preventing and treating osteoporosis, and particularly can be prepared into a medicament for promoting osteogenic differentiation or a medicament for preventing and treating osteoporosis or diseases caused by obesity. The invention not only provides a new application direction of the betaine, but also provides a new therapeutic drug and a new therapeutic approach for treating diseases caused by osteoporosis andor obesity.
Drawings
FIG. 1 shows the measurement of cell proliferation and cell viability of hAD-MSCs and hUC-MSCs treated with betaine at various concentrations by the CCK-8 method. Different concentrations of betaine: b _0mM, B _10 mM, B _40 mM, B _80mM, B _120 mM, cell viability for treatment time 24h to 96 h. Wherein A is the morphological characteristics of hUC-MSCs and B is hAD-MSCs with the scale bar of 500 μm. OD was measured at a wavelength of 450 nm.
FIGS. 2A-2E are graphs showing the effect of betaine on osteogenic and adipogenic differentiation of hAD-MSCs. Where 2A was ALP staining after 7 days (scale bar =500 μm); 2B was ARS staining after 3 weeks (scale bar =100 μm); 2C is oil red O staining (scale bar =100 μm) after 12 days, 2D is the area of staining quantified with ImageJ. 2E is adipogenic and osteogenic markerExpression of (2). Data were expressed as mean ± SD (n =3; * p <0.05， ** p <0.01)。
FIGS. 3A-3J are transcriptome analyses of betaine-treated MSCs, wherein 3A is PCA analysis of the different concentrations of betaine-treated groups; fig. 3B is a volcanic plot of B _10 mM vs B _0mm differentially expressed gene and (C) B _80mM vs B _0mm differentially expressed gene, where purple and cyan represent | FC | >1.4 and p.adj < 0.05.3D is real-time fluorescent quantitative PCR analysis of gene expression in hAD-MSCs treated with betaine at different concentrations, all values were normalized with GAPDH; 3E is an ELISA method for measuring the expression of IL6 in hAD-MSCs treated with or without betaine; 3F is the overlap between B _10 mM and B _0mM differentially expressed genes (adj. P value <0.05 and log2 FC > 1.4) and B _80mM and B _0mM differentially expressed genes; 3G is the study of biological processes of B _10 mM vs B _0Mm GO terms using DEGs; 3I is the biological process of GO terms using DEGs to study B _80mM vs B _0mM, the size of the circle is proportional to the number of genes in the class, reflecting differential expression, and the color represents p.adj.;3H and 3J are KEGG analyses relating to DEGs, with the ordinate representing the path name, the abscissa representing GeneRatio, and the size and color of the dots representing the number of differentially expressed genes in the path and p.adj.
Fig. 4 is a graph of the effect of betaine on osteogenic differentiation of hUC-MSCs, where a is ALP staining (scale bar =500 μm) after 7 days, B is alizarin red staining (scale bar =100 μm) after 3 weeks, and the stained area (C) was quantified with ImageJ. Data are in mean ± SD (n = 3;. P <0.05, p < 0.01).
FIGS. 5A-5I are transcriptome analyses of betaine-treated hUC-MSCs. Wherein 5A is PCA analysis of different concentrations of betaine; 5B is a volcano plot of B-10 mM vs B _0mM differentially expressed gene; 5C is a volcano plot of B _80mM vs B _0mm differentially expressed genes, where orange and blue represent | FC | >1.4 and p.adj < 0.05; FIG. 5D is a real-time fluorescent quantitative PCR analysis; FIG. 5E shows the ELISA detection of IL6 expression in hUC-MSC treated with different concentrations of betaine; FIG. 5F is a biological process using DEGs to study the B _10 mM vs B _0mM GO term; fig. 5H is a biological process of studying GO terms at B _80mM vs B _0mm using deg, the size of the circle being proportional to the number of genes of this class, reflecting differential expression, and the color representing p.adj.; fig. 5G and 5I are KEGG analyses associated with deg, with path names on the ordinate, geneRatio on the abscissa, and the size and colour of the dots representing the number of differentially expressed genes in the path and p.adj.
Detailed Description
In order to facilitate understanding of the technical solution of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples. Unless otherwise specified, all biochemical reagents used in the examples are commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art. The statistical analysis experiment of the invention is repeated for 3 times, all the experiments are independently repeated for at least 3 times, and the average value is represented as +/-standard deviation, and the statistical significance is that p is less than 0.05.
Example 1 cell culture.
Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are provided by the biotechnological biomedical (Shenyang) group, inc. P5 frozen adipose-derived stem cells (hAD-MSCs) were obtained from the first subsidiary hospital of the general Hospital of the liberation military. The hUC-MSCs and hAD-MSCs were thawed in a 37 ℃ water bath for 1-2 minutes and the cryoprotectant was removed by dropwise dilution with medium (Gbico, USA) supplemented with 10% fetal bovine serum (Excell, australia) and 1% penicillin (1000 Units/ml)/streptomycin (10000. Mu.g/ml) Dulbecco's modified eagle. The sample was centrifuged at 1000 rpm for 5 minutes and the resulting particles were suspended in complete medium at 37 ℃ plus 5% humidified CO 2 And (5) culturing. hUC-MSCs of 3-7 generations (P3-P7) and hAD-MSCs of 5-11 generations (P9-P11) were used.
Example 2 morphological characteristics and cytotoxicity assays.
Cell Counting Kit-8 (Dojindo, japan) was used to determine the cytotoxic effects of betaine on hUC-MSCs and hAD-MSCs. Briefly, hUC-MSCs (5000 cells/well, P7) and hAD-MSCs (5000 cells/well, P11) were plated overnight in 96-well plates, and 24h, 48 h, 72h and 96h were cultured with different concentrations of betaine (10 mM, 40 mM,80 mM, 120 mM), respectively, each set of 5 parallel wells. Thereafter, 10. Mu.L of CCK-8 buffer was added to each well at 37 ℃ and 3h was dark-cultured. The absorbance was measured at a wavelength of 450 nm.
Example 3 osteogenic differentiation.
To determine osteoblast differentiation in vitro, hUC-MSCs at P8 were plated on 12-well plates (1.0X 10) 5 Cells/well), hAD-MSCs at P12 were plated in 12-well plates (1.0X 10) 5 Cells/well). At 80% confluence, osteogenic medium (α -MEM, gibco, USA), 100 μ g/ml L (+) -ascorbic acid (Sigma-Aldrich, USA) and 5 mM β -disodium glycerophosphate hydrate (Sigma-Aldrich, USA), 10% fetal bovine serum, 1% P/S) were mixed with various concentrations of betaine (10 mM,80 mM).
Example 4 ALP and alizarin Red staining.
ALP staining was performed 7 days later using BCIP/NBT alkaline phosphatase color kit (Beyotime, china) for the early osteogenic differentiation phase. Briefly, cells were fixed with 4% paraformaldehyde (Bioss antibody, china) for 20 min and washed 3 times with PBS. Thereafter, the cells were stained with ALP staining solution in the dark for 15 minutes (hAD-MSCs) or 24 hours (hUC-MSCs). To investigate the effect of betaine on mineralization, alizarin Red S (Solarbio, china) staining was performed 21 days after the late stage of osteogenic differentiation. In summary, cells were fixed with 4% paraformaldehyde for 15 min, then stained with 0.2% alizarin red S solution for 30 min, and finally the stained cells were washed and observed.
Example 5 lipogenesis analysis.
hAD-MSCs (5 ×10 4 Cells/well) were seeded in 24-well plates at P10. At 90% fusion, hAD-MSCs were cultured in MesenCult-chamber adipogenic differentiation kit (human) (STEMCELL Technologies, canada) for 12 days, followed by oil Red O (Sangon Biotech, china). Mixing 0.5% oil red O stock solution with ddH 2 And mixing the O according to the proportion of 3:2 to prepare the oil red O working solution. Staining was simple, cells were fixed with 4% paraformaldehyde for 10min, ddH 2 Washing with O, treating with 60% isopropanol for 2 min, staining with oil red O for 10min, and washing to observe cells.
Example 6 RNA sequencing and sequencing data analysis.
P5 hUC-MSCs and P12 hAD-MSCs were plated on 60 mm plates, respectively, at 37 ℃ in 5% CO 2 Humidifying the cultured cells in the incubator until the cells are fused to about 80%, and respectively using betaine (0 mm, 10 m) with different concentrationsm,80 mm) for 72h, each set having 3 parallel hole samples. Collecting the cell pellet, extracting RNA, RNA Integrity Number (RIN)>7.0 RNA sequencing (GenePlus Co., ltd., china). All purified libraries were sequenced on the DNBSEQ-T7RS, genePlus Limited in Shenzhen, china, to obtain paired-end sequence reads of 150 bp.
The RNA-seq data analysis program includes raw read processing, filtering, clean read mapping and visualization. First, the quality of the raw data was checked with fastQC. Fastp filters out low quality bases. The washed sequence reads were then mapped to the human genome (GRCh 38, ensembles) using STAR. Gene expression levels were quantified using the featuous commands. The principal component analysis is done by Origin 2021 default parameters. Using an edge R package, modeling the genes by a negative binomial generalized log-linear model, and identifying significant Differential Expression Genes (DEGs). If the adjusted p-value is <0.05 and | FC | >1.4, the gene is considered to be differentially expressed between samples. GO and KEGG enrichment analyses were performed using the ClusterProfiler package.
Example 7 Real-Time PCR assay.
The remaining total RNA of RNA-seq was reverse transcribed to cDNA using HiScript III RT Supermix for qPCR (Vazyme Biotech, china) according to the manufacturer's protocol. RT-qPCR was performed using TB Green ™ Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, japan) according to the manufacturer's instructions. Experimental data generation Using the CFX96 Real-Time PCR detection System (Bio-RAD, USA).
Example 8 ELISA assay.
hUC-MSCs(5×10 4 Cells/well, P7) and hAD-MSCs (5X 10) 4 Cells/well, P10) were plated on 24-well plates, and the experimental groups were treated with different concentrations of betaine (10 mM,80 mM) for 72h, respectively, and cell supernatants were collected. ELISA assays were performed according to the protocol of the human IL-6 ELISA kit (NEOBIOSCIENCE, china). The OD detection wavelength was 450 nm.
Example 9 results of the experiment.
1. Betaine modulates the proliferation of hAD-MSCs in a dose-dependent manner.
In order to detect the potential toxicity of the betaine with different concentrations on hAD-MSCs and hUC-MSCs, the CCK-8 method is adopted, as shown in figure 1, the proliferation level of hAD-MSCs and hUC-MSCs is increased after the treatment of the betaine with low concentration (10 mM, 40 mM) and the inhibition effect is shown after the treatment of the betaine with high concentration (80 mM, 120 mM). 120 The sublethal concentrations of hAD-MSCs and hUC-MSCs were still not achieved with mM betaine, indicating that these cells are highly tolerant to betaine as shown in FIGS. 1A and 1B.
2. Betaine promotes osteogenic differentiation of hAD-and hUC-MSCs.
To determine the effect of different concentrations of betaine on osteogenic differentiation of hAD-MSCs and hUC-MSCs, ALP and Alizarin Red S (ARS) staining were evaluated. ALP staining showed that 10mM betaine significantly increased the number of ALP positive cells after 7 days of differentiation as shown in fig. 2A, 2D and fig. 4 (a and C), ARS staining showed more plaque-calcified extracellular matrix after 21 days of differentiation as shown in fig. 2B, 2D, 4 (B and C), while 80mM betaine showed osteogenesis inhibitory effect, indicating that long-term use of high concentrations of betaine inhibited proliferation of hAD-MSCs and hUC-MSCs, consistent with CCK-8 assay results as shown in fig. 1A and 1B. As shown in FIG. 3E, the expression levels of OPN, RUNX-2 and OCN were up-regulated in the 10mM betaine-treated group compared to the control group.
3. Betaine inhibits adipogenic differentiation of hAD-MSCs.
To evaluate the adipogenic potential of hAD-MSCs in the control and betaine-treated groups, oil red O staining was performed. The decrease in lipid droplet number and size was accompanied by a significant decrease in the expression of adipogenic major genes such as PPAR γ, CEBP α, and FASN, indicating that betaine significantly inhibited adipogenic differentiation of hAD-MSCs (fig. 2C-2E). Since 80mM betaine-treated hUC-MSCs oil red O staining was weak, inhibition by betaine was difficult to observe (data not shown), consistent with the compromise in adipogenic differentiation potential of umbilical cord-derived MSCs. Previous studies showed that osteogenic and adipogenic differentiation of MSCs may be mutually exclusive, consistent with ALP, ARS and oil red O staining results for 10mM betaine treatment, but both osteogenic and adipogenic differentiation of hAD-MSCs was down-regulated in the 80mM betaine treatment group, as shown in fig. 2A-2D, suggesting that high concentrations of betaine may not only have inhibitory effects, but also be detrimental to cells. Therefore, 10mM should be the optimum concentration of betaine for hAD-MSCs and hUC-MSCs.
4. Transcriptome analysis showed a potential mechanism for betaine differentiation of hAD-MSCs.
To investigate the mechanism of betaine involvement in the differentiation of hAD-MSCs and hUC-MSCs, RNA-seq was performed with betaine in undifferentiated medium. Since betaine has a more significant effect on the adipogenic and osteogenic potential of hAD-MSCs than hUC-MSCs, we focused on the effect of betaine on hAD-MSCs for further study. Principal Component Analysis (PCA) showed separation of the control group from the betaine treatment group, with the first three PCs (PC 1, PC2 and PC 3) accounting for the 78.4% variation, as shown in fig. 3A. 10 There were 314 significantly Differentially Expressed Genes (DEGs), 74 genes up-regulated and 240 genes down-regulated in the mM and 80mM betaine treated groups, as shown in fig. 3F. PPARGC1A, IGF, SCD were down-regulated by 2.13, 1.56, 1.59 fold in the 10mM betain treated group, respectively, as shown in FIG. 3B, and FASN, IGF1, PDE3B, SCD, SOCS3 were down-regulated by 1.78, 2.89, 3.22, 2.93, 1.5 fold in the 80mM betain treated group, respectively, as shown in FIG. 3C. Several important cytokines involved in ECM breakdown and inflammation, such as MMP3, TGFB3, and CCL7, were down-regulated, as shown in table 1. The expression levels of 8 hAD-MSCs candidate genes corresponding to RNA-seq were determined by RT-qPCR and ELISA, as shown in FIGS. 3D and 3E.
TABLE 1 selection of genes differentially expressed by betaine-treated group of hAD-MSCs versus control group.
To study the betaine-induced global changes, gene Ontology (GO) Bioprocess (BP) and Kyoto Encyclopedia of Genes and Genes (KEGG) enrichment analysis was applied. The most enriched BP terms were shared extracellular matrix tissue in the dosing group. Ossification, adipocyte differentiation and bone salt deposition were also enriched as shown in fig. 3G and 3I. KEGG showed that the PI3K-Akt and MAPK signaling pathways were the most significant enriched signaling pathways in the 10mM betaine treatment. Cytokine-cytokine receptor interactions, ECM-receptor interactions, TGF- β signaling pathways, and cholesterol metabolism were also enriched for the administered group, as shown in figures 3H and 3J. The PI3K-Akt signaling pathway has previously been shown to play an important role in adipogenic and osteogenic differentiation. Studies have shown that extracellular matrix (ECM) plays an important role in controlling the balance between osteogenic and adipogenic differentiation of MSCs. Taken together, these results suggest that betaine regulates adipogenic and osteogenic differentiation of hAD-MSCs through PI3K-Akt signaling pathways, cytokine-cytokine receptor interactions, and ECM-receptor interactions.
In the invention, through experimental research, the betaine promotes the proliferation of hUC-MSCs and hAD-MSCs under low-concentration treatment, and the 10mM betaine inhibits lipogenesis and promotes osteogenesis, particularly hAD-MSCs. On the transcription level, the complex interaction among the PI3K-Akt signal pathway, the cytokine-cytokine receptor interaction and the ECM-receptor interaction participates in the regulation and control of the betaine on the differentiation of hAD-MSCs. After the hAD-MSCs are treated by betaine, the expression of NOX4 and PPARGC1A, PDE3B, SOCS is reduced, and previous researches show that the excessive expression of NADPH oxidase 4 (NOX 4) promotes the generation of ROS, and further promotes the generation of fat and the accumulation of intracellular lipid. PPARGC1A is a major regulator of adipogenesis, and the down-regulation of its expression is associated with decreased adipogenesis. PDE3B regulates insulin-induced glucose uptake, GLUT-4 translocation, which is phosphorylated by Akt to accelerate cAMP degradation, resulting in decreased lipogenesis. The binding of SOCS3 to JAK kinases and cytokine receptors inhibits STAT3 activation, leading to downregulation of RUNX2 and ALP expression in MSCs, consistent with betaine inhibition of adipogenic differentiation of hAD-MSCs. For hUC-MSCs, upregulation of several cytokines such as CXCL3, CXCL5, CXCL8, IL6, CCL20, IL24, IL36B, MMP were observed as shown in FIGS. 5B-5D, as was the enhancement of cell chemotaxis and cell differentiation obtained in the oxidized GO enrichment assay as shown in FIGS. 5F and 5H. KEGG enrichment analysis showed that deg was significantly enriched in cytokine-cytokine receptor interactions, as shown in fig. 5F and 5H, suggesting a potential mechanism for betaine to regulate differentiation of the hUC-MSCs.
BMPR1B was down-regulated at low and high concentrations of betaine treatment, and studies demonstrated that expression of BMPR1B increased significantly during early osteoblast differentiation, while expression of IL6 in the hhc-MSCs and hAD-MSCs showed an opposite trend during betaine administration, as shown in fig. 3D and 3E. FIGS. 5D and 5E illustrate that there is a complex association between the gene regulatory network and the phenotypic outcome. Experimental data provided by the name show that betaine promotes osteogenesis of mesenchymal stem cells of hUC-MSCs and hAD-MSCs and inhibits adipogenic differentiation of the mesenchymal stem cells when the drug is administrated at low concentration. These effects are mediated by the PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, and ECM receptor interactions. The invention provides technical support for betaine in preparation of medicines for treating diseases caused by osteoporosis and obesity.
Claims (10)
1. The application of betaine in preparing medicine for preventing and treating osteoporosis and/or obesity.
2. The application of betaine in preparing medicines for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells.
3. The betaine and mesenchymal stem cells are applied to the preparation of medicines for treating diseases caused by osteoporosis and/or obesity.
4. A method for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells, the method comprising treating mesenchymal stem cells with betaine.
5. The use as claimed in any one of claims 1 to 3 and the method as claimed in claim 4, wherein betaine is used at a concentration of 10mM.
6. The use according to any one of claims 1 to 3 and the method according to claim 4, wherein the promotion of osteogenic differentiation of mesenchymal stem cells increases the number of ALP positive cells and plaque calcified extracellular matrix during osteogenic differentiation of mesenchymal stem cells, up-regulates the expression levels of OPN, RUNX-2 and OCN proteins during osteogenic differentiation of mesenchymal stem cells; inhibiting the mesenchymal stem cell from differentiating into fat and reducing the expression level of PPAR gamma, CEBP alpha and FASN genes in the mesenchymal stem cell fat and fat differentiation process.
7. The use of any one of claims 1 to 3 and the method of claim 4, wherein the mesenchymal stem cells are human adipose-derived mesenchymal stem cells, hAD-MSCs, or human umbilical cord mesenchymal stem cells, hUC-MSCs.
8. A medicament for the prevention and treatment of diseases caused by osteoporosis and/or obesity, said medicament comprising betaine.
9. An agent for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells, wherein the agent comprises betaine.
10. The pharmaceutical composition for preventing and treating osteoporosis and/or obesity according to claim 8 and the pharmaceutical composition for promoting osteogenic differentiation of mesenchymal stem cells and/or inhibiting adipogenic differentiation of mesenchymal stem cells according to claim 9, further comprising other effective ingredients which are compatible with betaine to act synergistically; also comprises a pharmaceutically acceptable carrier and/or excipient; the dosage forms of the medicine include but are not limited to tablets, granules, capsules, dripping pills, sustained release preparations, oral liquid preparations and injections.
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