Soybean peptide SOP as well as preparation method and application thereof

Abstract

The invention discloses soybean peptide SOP as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The soybean peptide SOP is dodecapeptide and has the activity of promoting osteoblast proliferation, differentiation and mineralization, and the amino acid sequence of the soybean peptide SOP is VVELLKAFEEKF. The soybean peptide SOP has the beneficial effects that by improving the activity of TGF-beta1 in osteoblasts (improving the protein expression level of TGF-beta1), the soybean peptide SOP mediates phosphorylation of p38-MAPK and Smad2 / 3 signal channels and induces downstream multi-path regulation and control, so that the proliferation, differentiation and mineralization of the osteoblasts can be remarkably promoted; in-vivo tests prove that the soybean peptide SOP can significantly recover bone mineral density loss and behaviors of a zebrafish glucocorticoid induced osteoporosis (GIOP) model, has good osteogenesis promoting activity, and can be used as a potential osteogenesis component in functional food.

Description

Technical Field

[0001] The present invention relates to a polypeptide and a preparation method and application thereof, and in particular to a soybean peptide SOP and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] As a natural active protein derived from soybeans, soy protein has always been a hot topic among researchers in the field of nutrition. Many literatures have reported the functional activities of soy protein, such as regulating immunity, promoting intestinal flora balance, anti-oxidation, and anti-cancer.

[0003] In 2011, Chiagn et al. fed ovariectomized mice with soy milk fermented with lactic acid bacteria and found that soy milk fermented with lactic acid bacteria had a significant regulatory effect on trabecular and cortical bone loss after estrogen deficiency. This indicates that soy protein has osteogenic activity in vivo and is a natural active substance with the potential to treat osteoporosis.

[0004] However, the research on small molecule soybean peptides is relatively backward, which limits the further development and application of soybeans. In previous studies, our research group has found that fermented sour soybean milk has osteogenic activity in in vitro osteoblast experiments, and used ultrafiltration and metal ion chelation affinity chromatography to separate and purify peptides with calcium ion binding ability. Its amino acid composition was identified as VVELLKAFEEKF by LC-MS / MS, but its osteogenic mechanism is still unclear. Therefore, this project aims to provide scientific basis and theoretical foundation for the application of soybean peptides as a potential osteogenic component in functional foods by evaluating the osteogenic activity and osteogenic mechanism of soybean peptides in zebrafish larvae and pre-osteoblasts MC3T3-E1. Summary of the invention

[0005] The purpose of the present invention is to provide a soybean peptide SOP derived from soybean and having the activity of promoting osteoblast proliferation, differentiation and mineralization, as well as a preparation method and application of the soybean peptide SOP.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The soybean peptide SOP is a twelve-peptide having the activity of promoting osteoblast proliferation, differentiation and mineralization, and the amino acid sequence is VVELLKAFEEKF, as shown in SEQ ID NO: 1 in the sequence table.

[0008] The aforementioned soybean peptide SOP is used in the preparation of any one of a preparation for promoting osteoblast proliferation, a preparation for promoting osteoblast differentiation, and a preparation for promoting osteoblast mineralization.

[0009] The preparation method of the aforementioned soybean peptide SOP comprises the following steps:

[0010] (1) Soaking soybeans, adding water to grind the soybeans, removing the dregs, and obtaining soy milk;

[0011] (2) adding sucrose in an amount of 1 g / 50 mL to soy milk and sterilizing to obtain a soy milk culture medium;

[0012] (3) activating Lactobacillus plantarum to obtain seed liquid;

[0013] (4) inoculating Lactobacillus plantarum into a soy milk medium at an inoculum rate of 10%, fermenting at 37° C. for 12 h, and taking the fermentation liquid after 12 h of fermentation after the bacteria are stable for 5 generations to obtain sour soy milk;

[0014] (5) centrifuging the sour soybean milk in a high-speed refrigerated centrifuge, filtering the supernatant with a 0.45 μm filter membrane, adding the filtrate to the upper layer of a 3 kDa ultrafiltration centrifuge tube, centrifuging in a high-speed refrigerated centrifuge, collecting the lower layer sample and freeze-drying it to obtain a crude soybean peptide freeze-dried powder;

[0015] (6) Purify the crude lyophilized powder of soybean peptide using a polysaccharide gel G-15 chromatography column, using PBS as the eluent for two column volumes, and use a UV spectrophotometer to detect the absorbance of each tube of sample at 215 nm, 220 nm, and 280 nm, respectively. Collect the sample corresponding to the No. 4 absorption peak, desalt it with a dialysis bag, and freeze-dry it to obtain a pure lyophilized powder of soybean peptide with an amino acid sequence of VVELLKAFEEKF.

[0016] The benefit of the present invention lies in that the soybean peptide SOP provided by the present invention can significantly promote osteoblast proliferation, differentiation and mineralization by increasing the activity of TGF-β1 in osteoblasts (increasing the protein expression level of TGF-β1), thereby mediating the phosphorylation of p38-MAPK and Smad2 / 3 signaling pathways and inducing downstream multi-pathway regulation. In vivo experiments have confirmed that soybean peptide SOP can significantly restore bone density loss and behavior in the zebrafish glucocorticoid-induced osteoporosis (GIOP) model, has good osteogenic activity, and can be used as a potential osteogenic ingredient in functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The effect of different concentrations of SOP on the proliferation and cell cycle of MC3T3-E1 cells. (A) Effect of SOP (0.7, 7, 70 μM) on cell proliferation for 24, 48 and 72 hours. (B) MC3T3-E1 cells were treated with SOP at different doses for 24 hours and then collected and analyzed by flow cytometry. (C) Cell cycle distribution of MC3T3-E1 cells in G0 / G1, S and G2 / M phases after 24 hours of SOP treatment. The results are expressed as mean ± standard deviation (SEM), and statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n = 3-4. *p<0.05, **p<0.01; ***p<0.001, compared with the control group.

[0018] Figure 2 Effects of different concentrations of SOP on the differentiation and mineralization of MC3T3-E1 osteoblasts. (A) Effects of SOP (0.7, 7, 70 μM) treatment for 24 hours on ALP activity. (B) Effects of SOP (0.7, 7, 70 μM) treatment for 24 hours on OCN concentration. (C) ARS staining was used to detect the ability of cells to form calcified nodules after 7, 14, and 21 days of osteogenic induction. The images are the staining effects of 6-well plates. (D) ARS staining was used to detect the ability of cells to form calcified nodules after 7, 14, and 21 days of osteogenic induction. The microscope magnification was 100 times. (E) After decolorization with cetylpyridinium chloride in sodium phosphate, the absorbance was measured at 490 nm. Data are expressed as mean ± standard deviation (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n = 3. *p < 0.05; ***p < 0.001, compared with the control group.

[0019] Figure 3 The effect of SOP on the activation of TGF-β / Smad pathway in MC3T3-E1 cells. (A) and (B) Effects of different concentrations of SOP on the expression of TGF-β1 protein in MC3T3-E1 cells. (C) and (D) Effects of 70 μM SOP on the phosphorylation levels of Smad2 and Smad3 pathways in MC3T3-E1 cells after treatment for 0, 15, 30, 45, and 60 minutes, respectively. (E) and (F) Effects of different concentrations of SOP on the expression of Smad4 nuclear protein in MC3T3-E1 cells. (G) and (H) Effects of different concentrations of SOP on the expression of Runx2 protein in MC3T3-E1 cells. Data are expressed as mean ± standard deviation (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n = 3. *p < 0.05; **p < 0.01, ***p < 0.001, compared with the control group.

[0020] Figure 4 is the effect of SOP on the activation of MAPK pathway in MC3T3-E1 cells. (A) and (B) Effects of 70 μM SOP on the phosphorylation levels of ERK, JNK, and p38 pathways in MC3T3-E1 cells within 24 h. Data are presented as mean ± standard deviation (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Sample size was n = 3, ***p < 0.001, compared with the control group.

[0021] Figure 5 SOP stimulates osteoblast differentiation by activating the Smad2 / 3 and p38-MAPK signaling pathways. (A) Effects of MAPK inhibitors (SB203580, SP600125, and U0126) and Smad2 / 3 inhibitor (SB431542) on SOP (70 μM) to enhance ALP activity in MC3T3-E1 cells. (B) Effects of p38 inhibitor (SB203580) and Smad2 / 3 inhibitor (SB431542) on SOP (70 μM) to enhance intracellular OCN concentration in MC3T3-E1 cells. Data are presented as mean ± standard deviation (SEM). One-way analysis of variance (ANOVA) was used for analysis, followed by Tukey's multiple comparison test (n=3). *p<0.05; ***p<0.001, compared with the control group, ns indicates no significance.

[0022] Figure 6 SOP induces intracellular signaling in MC3T3-E1 cells through the TβRI receptor. (A) and (B) Effects of TβRI inhibitor (SB525334) on SOP (70 μM)-induced enhancement of phosphorylation levels of Smad2 and Smad3 pathways in MC3T3-E1 cells. (C) and (D) Effects of TβRI inhibitor (SB525334) on SOP (70 μM)-induced enhancement of phosphorylation levels of p38 pathway in MC3T3-E1 cells. Data are expressed as mean ± standard deviation (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, n = 3. *p < 0.05; **p < 0.01, compared with the control group.

[0023] Figure 7SOP promotes MC3T3-E1 cell differentiation by increasing the activity of TGF-β1. (A) and (B) Effects of 70 μM SOP and 1 ng / mL TGF-β1 on the phosphorylation levels of Smad2 and Smad3 pathways in MC3T3-E1 cells in α-MEM without FBS. (C) Effects of 70 μM SOP and 1 ng / mL TGF-β1 on ALP activity in MC3T3-E1 cells (24 h). (D) SOP promotes TGF-β1 activation by activating TGF / Smad and p38-MAPK pathways, thereby stimulating osteoblast differentiation. Data are expressed as mean ± standard deviation (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (n = 3). **p < 0.01; *p < 0.001, compared with the control group, ns indicates no significant difference.

[0024] Figure 8 is the anti-osteoporotic effect of SOP in the glucocorticoid-induced zebrafish osteoporosis model. (A) Fluorescence images of 7dpf zebrafish skulls treated with (a) 0.1% DMSO (control), (b) 25μM prednisolone (inducer), or co-administered with SOP (3.3μM, 10μM, 30μM) (ce), or 0.308μM alendronate (positive control) (f) at 96hpf. (B) Relative fluorescence intensity (RFI) of 7dpf zebrafish skulls. (C) Movement distance of zebrafish larvae after 96hpf after SOP intervention. (D) Swimming speed of zebrafish larvae after 96hpf after SOP intervention. n=6-10. Data are presented as mean ± standard deviation (SEM). ##p<0.01; ###p<0.001; compared with the control group, *p<0.05; **p<0.01; ***p<0.001, compared with the prednisolone group (Pre), ns means no significant difference. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] 1. Ferment soybeans and separate and purify soybean peptide SOP

[0027] Take 300g soybeans, soak them in ultrapure water for 12h, add 12 times the volume of ultrapure water to grind, filter out the dregs with 100-mesh gauze to obtain soybean milk. Take 300mL soybean milk and pour it into a 500mL conical flask, add 6g sucrose, seal the bottle mouth with a sealing film, place it in a vertical pressure steam sterilizer and sterilize it at 115℃ for 15min, cool it, and obtain soybean milk culture medium.

[0028] Take out 1 mL of Lactobacillus plantarum from a -20°C refrigerator, inoculate it into 10 mL of MRS broth medium, and culture it in a 37°C electric constant temperature incubator. After 24 hours, take out 10 mL of the activated bacterial liquid and inoculate it into 100 mL of MRS broth medium and culture it for 12 hours to obtain seed liquid.

[0029] The plant lactobacillus was inoculated into the soybean milk medium at an inoculation rate of 10%, and fermented at 37° C. for 12 hours. After the bacteria were stable for 5 generations, the fermentation liquid after 12 hours of fermentation was taken to obtain sour soybean milk.

[0030] The sour soybean milk was placed in a high-speed refrigerated centrifuge and centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was filtered with a 0.45 μm filter membrane, and the filtrate was added to the upper layer of a 3 kDa ultrafiltration centrifuge tube. The filtrate was centrifuged at 4°C and 4000 rpm for 10 min in a high-speed refrigerated centrifuge. The lower layer sample (molecular weight <3 kDa) was collected and freeze-dried to obtain a crude soybean peptide freeze-dried powder.

[0031] The crude lyophilized powder of soybean peptide was purified by using a dextran gel G-15 chromatography column (which can separate small molecule polypeptides of 100-1500Da). The specification of the chromatography column was 16mm*70cm. PBS was used as the eluent for elution. The concentration of the loading solution was 50mg / mL, the loading volume was 1mL (flow rate 0.08mL / min), the eluent flow rate was 0.16mL / min, and the elution was 19h (two column volumes). The automatic collector was set to collect one tube of sample every 19min. The volume of the sample collected in each tube was 3.04mL, and a total of 60 tubes were collected.

[0032] Use an ultraviolet spectrophotometer to detect the absorbance of each tube of sample at 215nm, 220nm, and 280nm, collect the samples corresponding to each absorption peak, desalt each sample using a dialysis bag with a molecular weight cutoff of 100-500kDa, freeze-dry, and obtain pure soybean peptide freeze-dried powder.

[0033] The UPLC-Q-TOF method identified that the main component of the pure lyophilized powder of soybean peptide corresponding to the absorption peak No. 4 was the dodecapeptide VVELLKAFEEKF (SEQ ID NO: 1), hereinafter referred to as soybean peptide SOP.

[0034] 2. Study the effects of soybean peptide SOP on osteoblast proliferation, differentiation and mineralization

[0035] 1. Detection of the effect of soybean peptide SOP on osteoblast proliferation

[0036] The cell proliferation was detected by MTT assay. The MC3T3-E1 osteoblast precursor cell line was seeded at a density of 3 × 10 3 / well, different concentrations (0.7μM, 7.0μM, 70μM) of soybean peptide SOP were added at 100μL / well, and co-incubated at 37°C. The absorbance at 570nm was measured by an ELISA reader at 24h, 48h, and 72h of co-incubation. 4-5 groups of parallel wells were set for each sample. The results of the proliferation test of MC3T3-E1 osteoblast precursor cell line are shown in Figure 1 (A) By Figure 1 (A) It can be seen that soybean peptide SOP promotes osteoblast proliferation in a dose- and time-dependent manner. In addition, cell proliferation can be controlled by changing the phase of the cell cycle. The cell cycle was measured using a cell cycle and apoptosis kit. Figure 1 (B) and Figure 1 (C) It can be seen that soybean peptide SOP promotes cell proliferation by accelerating the cell cycle process.

[0037] 2. Detection of the effect of soybean peptide SOP on osteoblast differentiation

[0038] The differentiation of osteoblasts is crucial to the anabolism of bone. During the differentiation of osteoblasts, alkaline phosphatase (ALP) and osteocalcin (OCN) are the characteristic phenotypic markers of the early and late stages of differentiation, respectively. The activity of ALP during differentiation was determined using an ALP kit, and the secretion of OCN during differentiation was determined using a mouse osteocalcin ELISA kit.

[0039] 1) Detection of the effect of soybean peptide SOP on the activity of ALP, a marker of early differentiation

[0040] MC3T3-E1 osteoblast precursor cells were cultured at 2×10 5 The cells were inoculated at a density of 100 μg / well in a 6-well plate, cultured in complete medium for 2 days, then cultured in differentiation medium for 4 days, and then cultured in serum-free medium on the 7th day. 3 mL of soybean peptide SOP of different concentrations (0.7 μM, 7.0 μM, 70 μM) were added, respectively. After 24 hours of incubation, the cells were broken and the protein was extracted by centrifugation. The ALP activity of the protein was determined using an ALP kit. Three parallel wells were set for each sample.

[0041] The ALP activity test results of the protein are shown in Figure 2 (A) By Figure 2 (A) It can be seen that soybean peptide SOP can significantly increase the activity of ALP in osteoblasts, and the activity increases with increasing doses.

[0042] 2) Detect the effect of soybean peptide SOP on the secretion of OCN, a marker of late differentiation

[0043] MC3T3-E1 osteoblast precursor cells were cultured at 2×10 5The cells were inoculated at a density of 100 μg / well in a 6-well plate, cultured in complete medium for 2 days, then cultured in differentiation medium for 11 days, and then cultured in serum-free medium on the 14th day. 3 mL of soybean peptide SOP at different concentrations (0.7 μM, 7.0 μM, 70 μM) were added, respectively. After 24 h of incubation, the supernatant was collected by centrifugation, and the OCN content was determined using a mouse osteocalcin ELISA kit. Three parallel wells were set for each sample.

[0044] The relative amount of OCN in the supernatant was determined as shown in Figure 2 (B) By Figure 2 (B) It can be seen that soybean peptide SOP can significantly promote the secretion of OCN by osteoblasts, and the secretion increases with the increase of dosage.

[0045] 3) Detect the effect of soybean peptide SOP on the late mineralization of osteoblasts

[0046] MC3T3-E1 osteoblast precursor cell line was cultured with differentiation medium containing ascorbic acid (50 μg / mL) and β-glycerophosphate (10 mM), and the cells were treated with 3 mL of soybean peptide SOP at different concentrations (0.7 μM, 7.0 μM, 70 μM) for different days (7 days, 14 days, 21 days), during which the differentiation medium was replaced every 3 days, and the same amount and concentration of soybean peptide SOP were added. After the culture, the cells were specifically stained with Alizarin Red and photographed under a microscope, and then the cells were decolorized with cetylpyridinium chloride, and finally the absorbance at 490 nm was measured with an enzyme reader. Three sets of parallel wells were set for each sample.

[0047] The cell staining results are shown in Figure 2 (C) and Figure 2 (D), the absorbance measurement results after cell decolorization are shown in Figure 2 (E) By Figure 2 (E) It can be seen that as the treatment time increases, soybean peptide SOP can significantly promote osteoblast mineralization, and the effect becomes more prominent as the dose increases.

[0048] 3. Study on the mechanism of action of soybean peptide SOP on osteoblasts

[0049] 1. Analysis of activation of TGF-Smad pathway in MC3T3-E1 cells induced by SOP

[0050] TGF-β regulates osteoblast growth and development mainly by initiating intracellular signal transduction and activating the Smad2 / 3 signaling pathway. In order to prove the role of TGF-β / Smad pathway in SOP-induced osteoblast differentiation, the activation of Smad2 / 3 signaling pathway by SOP at different times was detected. Figure 3 (A) and Figure 3(B) shows that the protein expression level of TGF-β1 in the 70 μM SOP treatment group increased significantly by 64.51% compared with the control group.

[0051] (p<0.01), indicating that TGF-β may be a potential pathway for SOP to promote osteoblast differentiation. Figure 3 (C) and Figure 3 (D) shows that 70 μM SOP treatment significantly increased the phosphorylation of Smad2 and Smad3 in MC3T3-E1 cells (p<0.001). Since the different activation of Smad2 and Smad3 is caused by the interaction with a unique set of proteins on the TGF-β receptor, this result may be due to the different activation patterns of Smad2 and Smad3. After 60 minutes of 70 μM SOP treatment of MC3T3-E1 cells, the nuclear translocation of Smad4 was significantly increased by 54.44% ( Figure 3 (E) and Figure 3 (F), p<0.05). Osteoblast differentiation was significantly affected by Runx2, a downstream target of TGF-β signaling. Figure 3 (G) and Figure 3 (H) shows that Runx2 phosphorylation significantly increased by 58.08% (p<0.001) after MC3T3-E1 cells were treated with 70 μM SOP for 24 hours. These results indicate that Smad2 and Smad3 can be activated by SOP and form complexes with Smad4, which accumulate in the nucleus to regulate downstream gene expression, including Runx2.

[0052] 2. Analysis of activation of MAPK pathway in MC3T3-E1 cells induced by SOP

[0053] The MAPK pathway is an independent pathway for TGF-β signaling and is involved in bone formation. Many classic osteogenic active substances regulate the expression of osteogenic genes by activating the MAPK pathway to promote osteoblast differentiation. Therefore, we studied the phosphorylation status of ERK, JNK, and p38 under SOP stimulation. Figure 4 (A) and Figure 4As shown in (B), 70 μM SOP treatment significantly increased the phosphorylation of ERK, JNK, and p38 pathways, but the activation time was different (p<0.001). First, the phosphorylation level of ERK reached a peak at 3 hours of incubation with SOP (5.42 times the ERK phosphorylation level at 0 hours), and then slowly decreased until 24 hours, indicating that SOP can quickly and continuously activate the ERK pathway. After SOP treatment, the phosphorylation of p38 and JNK pathways was transiently activated, reaching a peak at 3 hours (2.73 times and 1.82 times the JNK and p38 phosphorylation levels at 0 hours, respectively), and then quickly returned to the basal level, indicating that the activation of p38 and JPK pathways by SOP is transient, and the main action phase may be concentrated in 0-3 hours. These data suggest that SOP has the potential to promote osteoblast differentiation by activating the MAPK pathway.

[0054] 3. SOP promotes the differentiation of MC3T3-E1 cells by activating Smad2 / 3 and p38 pathways

[0055] To explore the signal transduction pathways by which SOP promotes osteoblast differentiation of MC3T3-E1 cells, MAPK inhibitors (SB203580, SP600125, and U0126) and Smad2 / 3 inhibitor (SB431542) were used to prevent the activation of p38, JNK, ERK, and Smad2 / 3 pathways. Figure 5 (A) shows that there was no significant difference in ALP activity between the group containing the inhibitor alone and the control group (p>0.05). In addition, in the group containing SOP and inhibitors, ALP activity stimulated by SOP was not affected by treatment with JNK inhibitors and ERK inhibitors ( Figure 5 (A)). However, the addition of p38 inhibitor and Smad2 / 3 inhibitor significantly reduced the ALP activity increased by SOP ( Figure 5 (A), p<0.001). Similarly, when treated with p38 inhibitors and Smad2 / 3 inhibitors, respectively, the phosphorylation of p38 pathway and Smad2 / 3 pathway was blocked, resulting in an almost complete disappearance of the promoting effect of SOP on OCN content in MC3T3-E1 cells ( Figure 5 (B)). The results showed that blocking the p38 and Smad2 / 3 pathways affected the signal transduction of SOP in osteoblasts, resulting in the restoration of ALP activity and OCN content to basal levels, indicating that SOP may synergistically promote osteoblast differentiation through the p38 MAPK pathway and the Smad2 / 3 pathway. At the same time, this is the first time that a naturally derived small molecule peptide has been found to stimulate osteoblast differentiation through this pathway.

[0056] 4. Analysis of signal transduction in MC3T3-E1 cells induced by SOP via TβRI receptor

[0057] To verify whether TGF-β1 receptor protein is involved in the process of SOP promoting osteogenic differentiation, cells were treated with SOP in the presence or absence of TβRI inhibitor SB431542, and intracellular signal transduction was detected by Western blotting. The protein band results showed that the inhibitor SB431542 significantly inhibited the phosphorylation of Smad2 / 3 and p38 stimulated by SOP ( Figure 6 , p<0.05). In addition, these data also indicate that TβRI is involved in the osteoblast differentiation process induced by SOP.

[0058] 5. Study on the effect of SOP on the differentiation of MC3T3-E1 cells by promoting the activity of TGF-β1

[0059] In order to demonstrate how SOP acts on MC3T3-E1 cells to exert its osteogenic activity, cells were treated with serum-free medium to exclude the interference of TGF-β1, and the phosphorylation level of Smad2 / 3 and ALP activity were detected. The protein band results showed that TGF-β1 significantly improved the phosphorylation of Smad2 / 3 ( Figure 7 (A) and Figure 7 (B), p<0.01). In addition, compared with the group treated with TGF-β1 alone, the SOP and TGF-β1 treatment groups significantly stimulated the phosphorylation of Smad2 / 3 ( Figure 7 (A) and Figure 7 (B), p<0.01). However, compared with the control group, the SOP treatment group could not significantly promote the phosphorylation of Smad2 / 3 (p>0.05). At the same time, the data of ALP activity also showed the previous results. The SOP and TGF-β1 treatment groups significantly stimulated ALP activity ( Figure 7 (C), p<0.001) These results indicate that SOP alone did not exert its osteogenic activity and that its promotion of osteoblast differentiation was achieved by promoting the activity of TGF-β1 ( Figure 7 (D)).

[0060] IV. Evaluation of the in vivo osteogenesis-promoting effect of soybean peptide SOP

[0061] 1. Animal Model

[0062] Modeling of the zebrafish glucocorticoid-induced osteoporosis (GIOP) model: Select appropriate 72hpf embryos and incubate them with water containing 25μM prednisolone and SOP or ALN for 4 days, injecting fresh water every day. Finally, different groups of 7dpf zebrafish larvae were used for subsequent experiments. All zebrafish experiments were divided into six groups: zebrafish embryos were treated with 0.1% DMSO (control group), 25μM prednisolone (model) or co-incubated with SOP (3.3, 10, 30μM) or 0.308μM ALN (positive control).

[0063] 2. Experimental treatment methods

[0064] The experiment was set up in six groups: zebrafish embryos were treated with 0.1% DMSO (vehicle), 25μM prednisolone (model) or co-incubated with soybean peptide SOP (3.3, 10, 30μM) or 0.308μM ALN (positive control). Using prednisolone-induced glucocorticoid-induced osteoporosis (GIOP) zebrafish model, soybean peptide and prednisolone were simultaneously treated with zebrafish larvae until 7dpf, and then the changes in zebrafish bone density were detected by fluorescence microscopy after staining with the specific fluorescent dye calcein; the zebrafish behavior monitor was used to analyze the changes in the swimming speed and distance of zebrafish.

[0065] 3. Effect evaluation

[0066] Evaluation methods: Calcein staining and behavioral analysis: Calcein is a fluorescent dye that binds to calcified bone matrix. The bone mineral density of GIOP zebrafish was evaluated by measuring the fluorescence intensity of their skulls. Calcein staining and observation and photography were performed under a fluorescent inverted microscope, and the relative fluorescence intensity (RFI) of each zebrafish skull mass was calculated using the ImageJ tool. Zebrafish behavioral analysis: The zebrafish behavioral analysis system was used to analyze the changes in the swimming distance of zebrafish to evaluate whether the behavioral ability of zebrafish has improved. The time was set to 6 minutes, preparation time: sampling time = 1:1, and the ambient light intensity was set to 100%. The environment was kept quiet during the period.

[0067] 4. Conclusion

[0068] Figure 8 (A) shows representative images of zebrafish skulls at 7 dpf, and Figure 8 (B) The relative fluorescence intensity (RFI) of skull mass was quantified. The fluorescence intensity of the zebrafish skull in the model group was significantly reduced by 83.32% compared to the vehicle control group (p<0.001). When treated with 0.308 μM ALN, the reduction in fluorescence intensity was significantly reversed by 308.44% (p<0.001), indicating that the GIOP model was successful. The bone mass RFI increased significantly by 130.57%, 151.50%, and 394.39%, respectively (p<0.01). These findings indicate that SOP can significantly reverse prednisolone-induced bone density loss. Figure 8 (C) and Figure 8As shown in (D), prednisolone significantly reduced the effective travel distance and average swimming speed of zebrafish compared with the vehicle group. 0.308μM ALN showed a reversal of the decrease in travel distance and swimming speed compared with the model group. At the same time, 30μM SOP significantly increased the total distance from 174.667 mm to 404.391 mm and increased the average swimming speed from 0.238 mm / s to 0.540 mm / s in 10 min compared with the model group ( Figure 8 (C) and Figure 8 (D), p<0.05). These data suggest that SOP can partially reverse the lack of effects of GIOP in zebrafish. These findings suggest that SOP can reverse the bone loss and behavioral defects in GIOP zebrafish.

[0069] This study examined the osteogenic activity of SOP in vitro and in vivo and explored its differentiation mechanism in MC3T3-E1 cells. SOP is a bioactive peptide extracted from soy yogurt and consists of twelve amino acids. It can promote osteoblast proliferation by changing the cell cycle and stimulate osteoblast differentiation and mineralization. In addition, SOP stimulates osteoblast differentiation by activating the Smad2 / 3 pathway and non-Smad pathway p38 MAPK through activating TGFβ1. This is the first time that a naturally derived small molecule peptide has been found to stimulate osteoblast differentiation by increasing TGFβ1 activity and inducing downstream multi-pathway regulation. In addition, SOP can reverse the bone loss and behavioral decline in GIOP zebrafish.

[0070] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Soybean peptide SOP, characterized in that: The soybean peptide SOP is a dodecapeptide having the activity of promoting osteoblast proliferation, differentiation and mineralization, and the amino acid sequence is VVELLKAFEEKF, as shown in SEQ ID NO: 1 in the sequence table.

2. Use of the soybean peptide SOP according to claim 1 in the preparation of a preparation for promoting osteoblast proliferation.

3. Use of the soybean peptide SOP according to claim 1 in the preparation of a preparation for promoting osteoblast differentiation.

4. Use of the soybean peptide SOP according to claim 1 in the preparation of a preparation for promoting osteoblast mineralization.

5. The method for preparing soybean peptide SOP according to claim 1, characterized in that: The following steps are involved: (1) Soaking soybeans, adding water to grind the soybeans, removing the dregs, and obtaining soy milk; (2) adding sucrose in an amount of 1 g / 50 mL to soy milk and sterilizing to obtain a soy milk culture medium; (3) activating Lactobacillus plantarum to obtain seed liquid; (4) inoculating Lactobacillus plantarum into a soy milk medium at an inoculum rate of 10%, fermenting at 37° C. for 12 h, and taking the fermentation liquid after 12 h of fermentation after the bacteria are stable for 5 generations to obtain sour soy milk; (5) centrifuging the sour soybean milk in a high-speed refrigerated centrifuge, filtering the supernatant with a 0.45 μm filter membrane, adding the filtrate to the upper layer of a 3 kDa ultrafiltration centrifuge tube, centrifuging in a high-speed refrigerated centrifuge, collecting the lower layer sample and freeze-drying it to obtain a crude soybean peptide freeze-dried powder; (6) Purify the crude lyophilized powder of soybean peptide using a polysaccharide gel G-15 chromatography column, using PBS as the eluent for two column volumes, and use a UV spectrophotometer to detect the absorbance of each tube of sample at 215 nm, 220 nm, and 280 nm, respectively. Collect the sample corresponding to the No. 4 absorption peak, desalt it with a dialysis bag, and freeze-dry it to obtain a pure lyophilized powder of soybean peptide with an amino acid sequence of VVELLKAFEEKF.

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