Application of purple sweet potato pigment in the preparation of products for inducing neural differentiation of stem cells

Abstract

The present invention discloses the use of purple sweet potato pigment in the preparation of an induced stem cell neural differentiation product, which belongs to the field of biomedicine technology. The stem cell is a mesenchymal stem cell. The mesenchymal stem cell is a bone marrow mesenchymal stem cell. The neural differentiation is differentiation into acetylcholine neurons. The induced stem cell neural differentiation product includes a drug that induces stem cells to differentiate into neurons or a culture medium that induces stem cells to differentiate into neurons. The induction method is: pretreating stem cells with a growth factor mixture, and then adding purple sweet potato pigment to promote the differentiation of stem cells into neurons. The concentration of the purple sweet potato pigment is 50~100μg / mL. Through research, the present invention found that purple sweet potato anthocyanins can increase miR-223-3p, target Atg2b, inhibit the development of autophagy, and then induce BMSCs to differentiate into cholinergic neurons.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of purple sweet potato pigment in the preparation of products for inducing neural differentiation of stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells found in a variety of tissues, possessing robust self-renewal and multidirectional differentiation potential. MSCs are considered to have significant potential for neural regeneration and repair, with increasing research in the treatment of nerve injury and neurodegenerative diseases. Under specific conditions, MSCs can differentiate into nervous system cells such as neurons and glial cells, making neuronal differentiation a key research topic. However, stem cell differentiation is a complex process regulated by multiple factors. Autophagy, a key mechanism for cellular self-cleaning and metabolic homeostasis, plays a crucial role in stem cell differentiation. While moderate autophagy promotes stem cell growth and differentiation, excessive autophagy can inhibit their differentiation potential. Therefore, regulating autophagic activity has become a key area of ​​research for promoting neural differentiation of stem cells.

[0003] Purple sweet potato (Purple sweet potato) is a root vegetable rich in nutrients and bioactive ingredients. It belongs to the Convolvulaceae family and is an annual herbaceous plant. It is a premium sweet potato variety cultivated in Japan. Its purple-red flesh contains anthocyanins, known as purple sweet potato color (PSPC). Research on purple sweet potato began relatively early, around the mid-20th century. However, the structure of purple sweet potato has always been the focus of international research, with more recent studies focusing on its physiological functions and antioxidant activity. Japan has conducted some research on the extraction, identification, and physicochemical properties of PSPC. Chinese researchers Lu Guoquan et al. began systematic research on this pigment in 1996 and have made significant progress. It has been reported that acylation of pigment molecules can improve their stability. Since purple sweet potato pigment is acylated, it is more stable and has broad application prospects. Currently, most studies on purple sweet potato pigment focus on its antioxidant properties. For example, "The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associatedwith Restoration of NAD+ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice" (Xin Wang et al., Molecules 2017, 22,1315; doi:10.3390 / molecules22081315) reported that PSPC was used to treat hepatitis; "Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-a-mediated mitochondrial biogenesis signaling in mice" (Jun Lu et al., Free Radical Biology & Medicine) reported that PSPC was used to treat hepatitis. 52 (2012) 646-659) reported that PSPC alleviated kainate-induced cognitive deficits in mice by promoting the estrogen receptor-a-mediated mitochondrial biogenesis signaling pathway; however, there has been no report on the use of PSPC to induce neural differentiation of mesenchymal stem cells. Summary of the Invention

[0004] In response to the above-mentioned prior art, the present invention aims to provide the use of purple sweet potato pigment in the preparation of products that induce neural differentiation of stem cells. Through research, the present invention discovered that purple sweet potato anthocyanins can inhibit autophagy by increasing miR-223-3p, targeting Atg2b, and thereby inducing the differentiation of mesenchymal stem cells (MSCs) into cholinergic neurons, thus opening up a new application for purple sweet potato pigment.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides application of purple sweet potato pigment in preparing a product for inducing neural differentiation of stem cells.

[0007] Preferably, the stem cells are mesenchymal stem cells.

[0008] Preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells.

[0009] Preferably, the neural differentiation is differentiation into acetylcholinergic neurons.

[0010] Preferably, the product for inducing neural differentiation of stem cells includes a drug for inducing stem cells to differentiate into neurons or a culture medium for inducing stem cells to differentiate into neurons.

[0011] Preferably, the induction method is: pretreating stem cells with a growth factor mixture, and then adding purple sweet potato pigment to promote the differentiation of stem cells into neurons.

[0012] Preferably, the growth factor mixture comprises 50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF, and 100 μg / mL epidermal growth factor EGF.

[0013] Preferably, the concentration of the purple sweet potato pigment is 50-100 μg / mL.

[0014] Preferably, the differentiation time is 5 to 10 days.

[0015] Beneficial effects of the present invention:

[0016] (1) Through research, the present invention found that purple sweet potato anthocyanins can inhibit the development of autophagy by increasing miR-223-3p, targeting Atg2b, and then inducing bone marrow mesenchymal stem cells (BMSCs) to differentiate into cholinergic neurons.

[0017] (2) The present invention expands the application field of purple sweet potato anthocyanins, provides a potential new method for the clinical use of autologous stem cells to treat neurodegenerative diseases, and provides a theoretical basis for the clinical application of stem cells, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Study on the mechanism of PSPC inhibition of cellular autophagy, where (a) shows the effect of PSPC treatment on LC3 punctate aggregation in cells; green is GFP-LC3, red is MAP2, and the lower left corner is an enlarged image of LC3 in the cell pointed by the white arrow; (b) shows the number of cells with LC3 punctate aggregation; (c) shows the number of LC3 puncta in each cell; **p<0.05, *p<0.01, the above experiments are representative of three replicates;

[0019] Figure 2 : Interfering with cell autophagy can affect the neural differentiation process of BMSCs mediated by PSPC, where (a) shows the effect of inhibiting autophagy on the neuronal marker MAP2 and cell morphology, and (b) shows the effect of enhancing autophagy on the neuronal marker MAP2 and cell morphology;

[0020] Figure 3 : Autophagy negatively regulates PSPC-mediated neural differentiation of BMSCs; (a) shows the expression changes of neuronal markers ChAT, NSE, autophagy-related proteins, and mTOR signaling pathway-related proteins after 3-MA blocking autophagy or rapamycin induction of autophagy; (b) shows the statistical analysis results of the grayscale values ​​of ChAT and NSE protein bands; (c) shows the statistical analysis results of the grayscale values ​​of the autophagy-related proteins p62, beclin-1, and LC3 protein bands; (d) shows the statistical analysis results of the grayscale values ​​of the p-mTOR and p-4EBP1 protein bands in the mTOR signaling pathway; **p<0.05, *p<0.01, the above experiments are representative of three replicates;

[0021] Figure 4 :The miRNAs that change when PSPC induces BMSCs to differentiate into neural tissues;

[0022] Figure 5 : Effects of transfection with miR-223-3p mimics and inhibitors on ChAT mRNA;

[0023] Figure 6 : Effects of transfection with miR-223-3p mimics and inhibitors on Atg2b mRNA;

[0024] Figure 7 : Cell survival rate of BMSCs after treatment with grape seed anthocyanin and purple sweet potato pigment for 1, 2, and 3 days;

[0025] Figure 8PSPC induced MSCs to differentiate into neuron-like cells and increased the expression of the neuronal marker MAP2 protein; (a) shows the morphological changes of MSCs after treatment with PSPC and GSPE; (b) shows the expression of the neuronal marker microtubule-associated protein MAP2 by immunofluorescence; (c) shows the fluorescence intensity of MAP2; (d) shows the quantification of MAP2-positive cells; data are shown as mean ± SD, **p < 0.01, compared with the ctr group;

[0026] Figure 9 : Purple sweet potato pigment induces neural differentiation of MSCs; (a) is the mRNA level of nestin in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (b) is the mRNA level of MAP2 in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (c) is the mRNA level of NSE in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (d) is the protein band of MAP2 and NSE in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 8 days; *p<0.05, compared with the ctr group;

[0027] Figure 10 : Gene expression of neuronal subtype markers in mesenchymal stem cells treated with purple sweet potato pigment or grape seed anthocyanin for 6 days;

[0028] Figure 11 : After mesenchymal stem cells were treated with purple sweet potato pigment for 8 days, the protein level of cholinergic neuron marker ChAT increased; (a) is the protein band of cholinergic neuron marker ChAT in each treatment group; (b) is the protein level of cholinergic neuron marker ChAT in each treatment group;

[0029] Figure 12 Acetylcholine stimulates PSPC-treated cells to produce Ca 2+ Influx phenomenon; (a) is the change of acetylcholine added to BM-MSCs treated with PSPC observed under laser scanning confocal microscopy. As time goes by, the Ca 2+ The fluorescence intensity increased and gradually returned to the resting level (arrow-marked cells). The lower left corner indicates the time (in seconds) after the addition of neurotransmitters. (b) Effects of various neurotransmitters on Ca2+ expression in the CTR group. 2+ (c) Effects of various neurotransmitters on Ca concentration in the GSPE group 2+ concentration; (d) The effects of various neurotransmitters on Ca 2+ The arrows indicate the time of adding neurotransmitters, Ach is acetylcholine, GABA is γ-aminobutyric acid, L-Glu is L-glutamate, and DA is dopamine. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0031] As mentioned in the background technology section, although PSPC is a natural anthocyanin, the composition of anthocyanins from different sources varies significantly. Patent application number CN202410795740.2 discloses a method for preparing anthocyanin-loaded neural stem cell membrane vesicles and their use in drugs for preventing and treating spinal cord injury. Anthocyanins extracted from grapes are used to repair damaged neurons rather than induce stem cell differentiation. Compared with other anthocyanins extracted from strawberries, red cabbage, and perilla, PSPC has higher stability and stronger antioxidant capacity; research on PSPC is still in its early stages both domestically and internationally.

[0032] Based on this, the present invention aims to provide the use of purple sweet potato pigment in the preparation of products that induce neural differentiation of stem cells. PSPC can inhibit brain inflammation, reduce oxidative stress, and resist neuronal apoptosis, thereby improving cognitive ability in mice and effectively preventing the onset and progression of Alzheimer's disease.

[0033] Autophagy is a cellular self-eating phenomenon. It encapsulates intracellular organelles and proteins awaiting degradation to form autophagosomes, which then fuse with lysosomes to form autolysosomes, degrading the encapsulated contents, thereby maintaining cellular homeostasis and organelle renewal. Because abnormal protein accumulation or amyloidosis is a major pathogenic factor in neurodegenerative diseases, the regulatory role of autophagy in neurodegenerative diseases has become a research hotspot in recent years. Abnormal accumulation of autophagic vesicles in neurites from the brains of patients with Alzheimer's disease (AD) has been observed, and autolysosomes in AD exhibit abnormal acidification. Previous studies have shown that autophagy levels increase during the neuronal differentiation of MSCs, and inhibiting autophagy can inhibit neural differentiation. However, negative effects of autophagy on neural differentiation have also been reported. For example, retinoic acid can activate autophagy, inhibit mTOR activity, and induce neuronal differentiation in mouse neuroblastoma cells. These data reveal a dual regulatory role for autophagy in neural differentiation. MicroRNAs (miRNAs) are a class of non-coding, single-stranded RNA molecules approximately 18-25 nucleotides long, encoded by endogenous genes. They can bind complementary proteins to the 3' untranslated region (3'UTR) of target gene mRNAs, leading to fragmentation and degradation of the target mRNA. A growing body of research indicates that miRNAs are involved in neural differentiation, neural fate determination, and neural migration.

[0034] This study used PSPC to induce neuronal differentiation of mouse bone marrow mesenchymal stem cells (BMSCs) and identified the differentiation type. Using whole-transcriptome sequencing, the authors screened for miRNAs involved and identified their target genes, correlating them with autophagy. This study explored the molecular mechanism by which PSPC induces neural differentiation of BMSCs, providing a scientific basis for the discovery of new treatments for neurodegenerative diseases. The authors found that miR-223-3p expression is elevated during PSPC-induced neural differentiation of BMSCs. Overexpression of miR-223-3p promotes neural differentiation, while inhibition of miR-223-3p inhibits it, suggesting a crucial role for miR-223-3p in PSPC-induced neural differentiation of BMSCs. Further mechanistic studies revealed that miR-223-3p targets the autophagy-related gene Atg2b, a hypothesis supported by preliminary Q-PCR results. This suggests that purple sweet potato anthocyanins can elevate miR-223-3p, targeting Atg2b and inhibiting autophagy, thereby inducing BMSCs to differentiate into cholinergic neurons.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0036] The purple sweet potato pigment (PSPC) of the present invention was purchased from Qingdao Pengyuan Natural Pigment Research Institute (Qingdao, China).

[0037] Its components were studied in "The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD+ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice" (Xin Wang et al., Molecules 2017, 22, 1315; doi:10.3390 / molecules22081315):

[0038] High performance liquid chromatography analysis showed that the main components were acylated anthocyanins (>90%), including peonidin-3-O-(6-O-caffeoyl-2-O-β-D-glucopyranoside)-5-O-β-D-glucoside, peonidin-3-O-(2-O-(6-O-caffeoyl-β-D-glucopyranoside)-6-O-caffeoyl-β-D-glucopyranoside)-5-O-β-D-glucopyranoside, peonidin-3-O-(2-O-(6-O-feruloyl-β-D-glucopyranoside)-6-O-caffeoyl-β-D-glucopyranoside)-5-O-β-D-glucopyranoside and cyanidin-3-O-(6-O-p-coumaroyl)-β-D-glucopyranoside. The remaining components were other flavonoids.

[0039] Grape seed anthocyanins were purchased from Shandong Pingju Biotechnology Co., Ltd.

[0040] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0041] Example 1: Stem cell induction

[0042] Mouse bone marrow mesenchymal stem cells were added to α-MEM medium (α-MEM medium + 10% fetal bovine serum) containing growth factors (50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF, and 100 μg / mL epidermal growth factor EGF) and cultured in an incubator at 37°C for 3 days to obtain preconditioned stem cells. Purple sweet potato pigment was then added to a concentration of 75 μg / mL to induce differentiation of the mouse bone marrow mesenchymal stem cells, and cholinergic neurons were obtained after 8 days.

[0043] Example 2: Mechanism Exploration

[0044] To investigate whether autophagy is involved in PSPC-induced neural differentiation, second-generation BMSCs (mesenchymal stem cells, derived from mouse bone marrow) were digested and seeded into six-well plates. After one day of adherent culture, the cells were treated according to the following groups: a normal group (nor) cultured with α-MEM medium alone (same as in Example 1); a control group (ctr), i.e., a growth factor-induced group, cultured with α-MEM medium containing 10% fetal bovine serum and growth factors (same as in Example 1); and a purple sweet potato anthocyanin-treated group (PSPC): The control group's culture medium (α-MEM medium containing 10% fetal bovine serum and growth factors as in Example 1) was supplemented with varying concentrations of purple sweet potato anthocyanin aqueous solution. The concentration gradient of purple sweet potato anthocyanin was set at 50 μg / mL and 100 μg / mL. Immunofluorescence staining was performed 72 hours after treatment to detect the distribution and relative expression of microtubule-associated protein 2 (MAP2) and the autophagy-related protein LC3.

[0045] (1) PSPC negatively regulates cell autophagy, thereby inducing neural differentiation of BMSCs.

[0046] Depend on Figure 1 During PSPC-induced neuronal differentiation of BMSCs, the punctate distribution of LC3 decreased, gradually becoming diffuse, and the number of LC3 puncta in MAP2-positive cells decreased significantly. In the nor group, cells showed more LC3 puncta and almost no MAP2-positive cells, indicating high autophagic activity and low neural differentiation efficiency in this group. In the ctr group, LC3 puncta were slightly less abundant than in the nor group, with a small number of MAP2-positive cells, indicating slightly reduced autophagic activity and the generation of a small number of cholinergic neurons. In cells treated with 50 μg / mL and 100 μg / mL PSPC, the number of LC3 puncta was significantly reduced, and a large number of cells expressed MAP2, demonstrating that PSPC treatment reduces autophagic activity and enhances the efficiency of neural differentiation of BMSCs.

[0047] To further analyze the role of autophagy in PSPC-induced neural differentiation, second-passage BMSCs were digested and seeded into six-well plates. After one day of adherence, cells were pretreated with either 5 mM 3-methyladenine (3-MA), an autophagy inhibitor, or 10 nM rapamycin, an autophagy inducer, for two hours. Cells were then treated with the drugs according to the aforementioned groups. Morphological observation and immunofluorescence staining for MAP2 were performed 72 hours later. Protein samples were collected for immunoblot analysis. Since both 3-MA and rapamycin are soluble in DMSO, a control solvent was used. Cells were pretreated with an equal volume of DMSO to that of 3-MA and rapamycin, designated the "solvent control."

[0048] Depend on Figure 2 It can be seen that after pre-treating the cells with the autophagy inhibitor 3-MA, the MAP2 protein had a higher expression level, and the positive cell rate was higher than that of the normal control group. In particular, the cells in the PSPC group showed stronger red fluorescence, indicating that the expression of MAP2 protein increased significantly after inhibiting autophagy, suggesting that inhibiting autophagy can enhance the ability of PSPC to induce neural differentiation of stem cells. However, after pre-treating the cells with the autophagy inducer rapamycin, it was found that the intensity of red fluorescence was very low, indicating that the expression of MAP2 protein was very low after inducing autophagy, and the bone marrow mesenchymal stem cells did not successfully differentiate into neural cells. Observation of cell morphology showed that after 3-MA inhibited autophagy activity, the stem cells induced by PSPC all differentiated into typical neuron-like cells. Rapamycin-induced autophagy activity, on the contrary, restricted the differentiation of stem cells, and the cells remained long spindle-shaped. The above data confirm that PSPC induces neural differentiation of BMSCs by negatively regulating autophagy.

[0049] Western blot analysis was used to further analyze the effect of autophagy disruption on PSPC-induced neural differentiation of BMSCs. Three days after autophagy disruption, cells were lysed using RIPA buffer and total protein was extracted. Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher, A55864). Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with primary antibodies against ChAT (Proteintech, 20747-1-AP), NSE (Proteintech, 10149-1-AP), p62 (Cell Signaling Technology, 5114), beclin-1 (Cell Signaling Technology, 3495), LC3 (Cell Signaling Technology, 2775), p-2448 mTOR (Cell Signaling Technology, 2971), mTOR (Cell Signaling Technology, 2972), p-4EBP1 (Thermo Fisher, 700238), 4EBP1 (Thermo Fisher, MA5-32481), or β-actin (Proteintech, 66009-1-Ig) at 4°C overnight. After washing three times with PBS, secondary antibodies conjugated with horseradish peroxidase (goat anti-rabbit-HPR, Proteintech, SA00001-2 or goat anti-mouse-HRP, Proteintech, SA00001-1) were added and incubated for 1 h at 37°C. The relevant proteins were detected using ECL enhanced chemiluminescence reagent (Thermo Fisher, 32209).

[0050] Depend on Figure 3It was shown that 3-MA inhibited autophagy and accelerated PSPC-induced neural differentiation, whereas rapamycin had the opposite effect. In the vehicle control, which did not interfere with autophagy, PSPC treatment increased the expression of neural markers, while beclin-1 levels decreased, ubiquitin-binding protein (p62) levels increased, and the conversion of the autophagy-related protein LC3 was inhibited, indicating that PSPC-induced neural differentiation can reduce autophagy. p62 protein expression was significantly increased after PSPC and 3-MA treatment (p<0.01), while expression was decreased after rapamycin treatment. The expression of the autophagy-related protein beclin-1 and LC3B-I / II conversion increased after rapamycin treatment, confirming the function of autophagy inhibitors and inducers. Furthermore, the expression of the recombinant choline acetyltransferase (ChAT) protein was significantly increased after 3-MA and PSPC treatment compared with the vehicle control, while expression was significantly decreased after rapamycin treatment. The expression trends of neuron-specific enolase (NSE) and ChAT were consistent. NSE is an acidic protease unique to neurons and neuroendocrine cells. This indicates that inhibiting autophagy can increase the expression of neuronal markers, while inducing autophagy can reduce the expression of neuronal markers. Autophagy negatively regulates the PSPC-mediated neural differentiation process of BMSCs.

[0051] (2) miR-223-3p participates in PSPC-induced neural differentiation of BMSCs

[0052] To further explore the mechanism by which PSPC inhibits autophagy and induces neural differentiation of BMSCs, whole transcriptome sequencing was performed on bone marrow mesenchymal stem cells (BMSCs) treated with PSPC, focusing on miRNA changes. The researchers found that PSPC altered the expression of multiple miRNAs, including upregulating miR-146a-5p and miR-223-3p and downregulating miR-92a-5p, miR-222-5p, miR-152-5p, miR-148b-5p, and miR-148a-5p. RNA was then extracted from cells after 1, 2, and 3 days of PSPC treatment, and miRNA expression levels were measured.

[0053] Depend on Figure 4 It can be seen that PSPC can upregulate miR-223-3p and inhibit the levels of miR-92a-5p, miR-222-5p, miR-152-5p, miR-148b-5p and miR-148a-5p, and the results are consistent with the results of whole transcriptome sequencing.

[0054] Analysis of miRNAs revealed that miR-223-3p was the most upregulated gene. TargetScan and other software predicted that miR-223-3p may target the autophagy-related genes Atg2b (gene ID: 314415) and Atg9a (gene ID: 363254). Therefore, miR-223-3p was selected for further investigation in subsequent experiments. BMSCs were transfected with a miR-223-3p mimic and a miR-223-3p inhibitor (miR-223-3p mimic purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR10000892-1-5; inhibitor purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR20000892-1-5) in a concentration gradient manner to examine the effects of overexpression or silencing of miR-223-3p on PSPC differentiation.

[0055] BMSCs were seeded in 24-well plates, with 30,000 cells per well. The next day, transfection was performed as follows: In tube A, a miR-223-3p mimic or inhibitor was dissolved in 50 μL of Opti-MEM (reduced serum medium), gently pipetting 3-5 times to mix thoroughly, and incubate at room temperature for five minutes. Simultaneously, a control group was established: a non-miR-223-3p mimic and a control miR-223-3p inhibitor (control mimic purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR1N0000001-1-5; control inhibitor purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR2N0000001-1-5) were dissolved in Opti-MEM as described above. In tube B, 1 μL of Lipofectamine™ 3000 transfection reagent was dissolved in 50 μL of Opti-MEM, gently pipetting 3-5 times to mix thoroughly, and incubate at room temperature for five minutes. Mix the two and let them stand at room temperature for 20 minutes. Add 100 μL of the transfection complex to serum-depleted α-MEM medium (Thermo Fisher, 12561056) in the well plate. Shake the well plate to mix thoroughly and incubate in an incubator for 6 hours. Serum (NOR), growth factor (CTR, as in Example 1), and purple sweet potato pigment (PSPC, final concentration 50 μg / mL) were added to the cells in the group. Samples were collected after 72 hours.

[0056] Depend on Figure 5 It can be seen that the expression of ChAT increased after cells were transfected with miR-223-3p mimics, that is, the effect of PSPC in inducing neural differentiation was enhanced after transfection with miR-223-3p. However, ChAT decreased after transfection with inhibitors, and the miR-223-3p inhibitor blocked the induction effect of PSPC. The above results indicate that PSPC can induce BM-MSCs by enhancing the expression of miR-223-3p.

[0057] Differentiation towards neurons, and reducing the expression of miR-223-3p prevented the neural differentiation of BM-MSCs.

[0058] (3) miR-223-3p targets Atg2b

[0059] TargetScan and other target gene analysis software were used to predict the potential target genes of miR-223-3p, and it was found that miR-223-3p may target the autophagy-related gene Atg2b. After transfecting miR-223-3p mimics and inhibitors into BMSCs, the cells were grouped and treated with drugs (drug concentration was 50 μg / mL). The grouping was the same as that of the experiment (2) in Example 2 to detect the effect of Atg2b in this process. Figure 6 As can be seen, the expression level of Atg2b was very low after transfection with miR-223-3p mimics, while the expression level of Atg2b was significantly increased after transfection with miR-223-3p inhibitors. These results indicate that during PSPC-induced neural differentiation of BMSCs, miR-223-3p can target Atg2b to block the autophagy process, thereby enhancing the efficiency of PSPC-induced neural differentiation of BMSCs.

[0060] Test example

[0061] (1) Relative expression of MAP2 protein

[0062] The experiment was divided into blank control group, grape seed anthocyanin group, and purple sweet potato pigment group; the test concentration of the grape seed anthocyanin group and the purple sweet potato pigment group was 75 μg / mL, and an equal amount of water was added to the blank control group.

[0063] The second generation BMSCs were prepared into single cell suspension using α-MEM medium (Thermo Fisher, 12561056) containing 10% fetal bovine serum at a density of 1×10 5cells / mL, and the cells were seeded into 6-well plates. After the mesenchymal stem cells adhered, the original culture medium was removed and replaced with α-MEM medium containing the growth factors described in Example 1 and 10% fetal bovine serum. The differentiation induction experiment was then initiated according to the grouping described above. A blank control group (ctr) consisted of cells supplemented with the same volume of water as the purple sweet potato pigment. A grape seed anthocyanin group (GSPE) consisted of cells supplemented with a 75 μg / mL aqueous solution of grape seed anthocyanin. A purple sweet potato pigment-treated group (PSPC) consisted of cells supplemented with a 75 μg / mL aqueous solution of purple sweet potato anthocyanin. This was designated Day 0. Throughout the experiment, cell morphology was observed daily in each group and photographed under an inverted microscope. Differentiation medium was replaced every two days (blank control: α-MEM medium + 10% fetal bovine serum + growth factors described in Example 1; GSPE group: α-MEM medium + 10% fetal bovine serum + growth factors described in Example 1 + GSPE; PSPC group: α-MEM medium + 10% fetal bovine serum + growth factors described in Example 1 + PSPC). On days 1, 2, and 3, the cell status was detected by CCK-8 assay, and on day 3, the relative expression of MAP2 protein was detected by immunofluorescence staining.

[0064] according to Figure 7 and Figure 8 As shown in the results, grape seed anthocyanins and purple sweet potato pigments have good biocompatibility with MSCs, but only purple sweet potato pigments can increase the protein expression of neuronal marker MAP2 in BMSCs.

[0065] (2) The experimental groups were the same as above. Total RNA was extracted from BMSCs cultured for 3 and 6 days in each group. The expression of neural-specific markers such as nestin, neuron-specific enolase (NSE), and microtubule-associated protein 2 (MAP2) was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and semi-quantitative analysis was performed. RNA was reverse transcribed into cDNA, amplified by qPCR, and the Cq value of each gene in each group was analyzed. On the 8th day, cells were lysed with RIPA lysis buffer to extract total protein. Protein concentration was determined using a BCA (Bicinchoninic Acid) protein quantification kit (Thermo Fisher, A55864). The protein was transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). PVDF membranes were incubated with primary antibodies against MAP2 (Proteintech, 17490-1-AP), NSE (Proteintech, 10149-1-AP), or Nestin (Proteintech, 19483-1-AP) overnight at 4°C. After washing three times with PBS, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (Proteintech, SA00001-2) for 1 hour at 37°C. Proteins were detected using enhanced chemiluminescence (Thermo Fisher, 32209).

[0066] according to Figure 9 As shown in the results, compared with the control group and the grape seed anthocyanin-treated group, purple sweet potato pigment significantly increased the expression of neural differentiation-related genes and proteins, confirming that purple sweet potato pigment can induce BMSCs to differentiate into neurons.

[0067] (3) Experimental grouping was the same as above. Total RNA was extracted from BMSCs cultured for 6 days in each group. The expression of neural-specific markers such as acetylcholine (ChAT), glutamate receptor (GluR), N-methyl-D-aspartate receptor 1 (NMDA1), γ-aminobutyric acid receptor α1 (GABRA1), and dopamine receptor D2 (DRD2) was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and semi-quantitatively analyzed. RNA was reverse transcribed into cDNA, which was then amplified by qPCR, and the Cq value of each gene in each group was analyzed.

[0068] according to Figure 10 As shown, purple sweet potato pigment can significantly increase the markers of acetylcholinergic neurons, but has no obvious effect on the expression of other subtypes of neuronal markers, indicating that the type of neurons induced by purple sweet potato pigment-induced mesenchymal stem cell differentiation is acetylcholinergic neurons.

[0069] On day 8, cells were lysed using RIPA buffer to extract total protein. Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher, A55864). The protein was transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with an anti-ChAT primary antibody (Proteintech, 20747-1-AP) overnight at 4°C. After washing three times with PBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Proteintech, SA00001-2) for 1 hour at 37°C. Proteins were detected using enhanced chemiluminescence (Thermo Fisher, 32209). A calcium ion fluorescent probe (Fluo-4 AM) was used to assess the responsiveness of cells to neurotransmitters at a concentration of 75 μg / mL after day 8 of culture in each group. Cells were stimulated with the neurotransmitter acetylcholine, and real-time analysis was performed using confocal laser scanning microscopy.

[0070] according to Figure 11 and Figure 12 As shown, the acetylcholinergic neurons induced by purple sweet potato pigment have electrophysiological activity and are functional neurons.

[0071] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Use of purple sweet potato pigment in the preparation of an induced stem cell neural differentiation product; the stem cells are bone marrow mesenchymal stem cells; the neural differentiation is differentiation into acetylcholinergic neurons; the induced stem cell neural differentiation product comprises a drug for inducing stem cell differentiation into neurons or a culture medium for inducing stem cell differentiation into neurons; the induction method comprises: pretreating stem cells with a growth factor mixture, followed by adding purple sweet potato pigment to promote stem cell differentiation into neurons; the growth factor mixture comprises 50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF, and 100 μg / mL epidermal growth factor EGF; the concentration of the purple sweet potato pigment is 50-100 μg / mL.

2. The use according to claim 1, characterized in that The differentiation time is 5 to 10 days.

Images