Sfrp2 functional polypeptides and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to SFRP2 functional polypeptide and application thereof. The present application provides SFRP2 functional polypeptide and application thereof. The SFRP2 functional polypeptide (SFRP2-PP96) can reduce the proportion of Th17 subpopulation in CD4+ T cells, increase the proportion of Treg subpopulation; inhibit the differentiation of CD4+ T cells in induction to Th17 subpopulation, promote the differentiation to Treg subpopulation; inhibit experimental colitis of mice; inhibit the proliferation of Th17 in colon tissue, promote the proliferation of Treg; inhibit the expression of pro-inflammatory proteins in inflammatory colon tissue, promote the expression of anti-inflammatory proteins; promote the recovery of colitis by regulating the proportion of intestinal flora; promote the treatment effect of experimental periodontitis. And it is a polypeptide preparation, which has characteristics superior to traditional full-length proteins, and is convenient for subsequent clinical application and promotion.

Description

[0001] This application claims priority to Chinese Patent Application No. 202311786953.0, filed on December 22, 2023, entitled "SFRP2 Functional Peptide and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, specifically to SFRP2 functional peptides and their applications. Background Technology

[0003] Periodontitis is a complex chronic infectious disease caused by a variety of etiologies and contributing factors, typically appearing in early adulthood. Persistent inflammation of the periodontal tissues leads to deepening periodontal pockets, periodontal purulent discharge, periodontal bone loss, and tooth loosening and loss. Repairing periodontal bone defects is a major challenge in the field of regeneration. Mild to moderate periodontitis is generally controlled through non-surgical treatments and oral hygiene education. Severe periodontitis requires surgical treatment, but traditional guided bone regeneration and guided tissue regeneration techniques have limited and unstable regenerative effects, failing to achieve complete regeneration of periodontal tissues. Furthermore, complex immune responses exist in the periodontal tissues of periodontitis, including innate and adaptive immune responses designed to control pathogen invasion. However, dysregulation of the innate and adaptive immune systems may play a significant role in the etiology of periodontitis. Cytokines produced by the immune response regulate the interactions and cellular networks between macrophages, T cells, B cells, epithelial cells, and other cell types. Pro-inflammatory cytokines activate the immune defense while also activating osteoclasts and matrix metalloproteinases (collagenases); anti-inflammatory cytokines participate in the maturation, proliferation, and isotype conversion of B cells, thereby inhibiting the progression of periodontitis. Severe periodontitis may not be cured by traditional therapies, while immunotherapy can serve as an adjunct or alternative to traditional treatments. Immunotherapy has been shown to potentially modulate dysregulated immunity in autoimmune and inflammatory diseases. Current treatments for periodontitis mainly focus on periodontal scaling and root planing, sometimes combined with antibiotics to remove biofilms. However, these therapies often fail to completely eliminate periodontal pathogens, whose persistence continues to stimulate the host's immune response. In turn, the dysregulated host immune response leads to further spread of periodontal pathogens.

[0004] In the immune response to periodontitis, CD4 + T cells play an important role. Th17 cells act as CD4 cells... +A unique lineage of T cells, Treg cells promote the progression of periodontitis through the secretion of the key cytokine IL-17. Treg cells are regulated by the transcription factor Foxp3, which induces Treg cell differentiation and downregulates Th17 cell differentiation through STAT6. The dynamic balance between Th17 and Treg cells is an important regulatory factor in inflammation. To date, the reasons for the Th17 / Treg imbalance during periodontitis are not fully understood.

[0005] The Wnt / β-catenin signaling pathway plays a crucial role in tooth development and differentiation. However, its exact function in periodontitis remains controversial.

[0006] Secreted frizzled-related protein 2 (SFRP2), as an antagonist of the Wnt / β-catenin signaling pathway, has many advantages, including regulating osteogenic / dentigenic differentiation of mesenchymal stem cells, promoting tissue repair and regeneration, and regulating immune balance. However, traditional recombinant proteins have low purity, low yield, long production cycle, and high production cost. Furthermore, they cannot introduce non-natural amino acids or undergo terminal amidation, which limits their further clinical translation and application. Summary of the Invention

[0007] In view of this, the present invention provides SFRP2 functional peptides and their applications.

[0008] This invention provides a functional peptide of SFRP2 and its applications. The SFRP2 functional peptide (SFRP2-PP96) can reduce the proportion of Th17 subset in CD4+ T cells and increase the proportion of Treg subset; inhibit the induced differentiation of CD4+ T cells into Th17 subset and promote differentiation into Treg subset; inhibit experimental colitis in mice; inhibit Th17 proliferation in colonic tissue and promote Treg proliferation; inhibit the expression of pro-inflammatory proteins in inflamed colonic tissue and promote the expression of anti-inflammatory proteins; promote the recovery of colitis by regulating the proportion of intestinal flora; and enhance the therapeutic effect of experimental periodontitis. Furthermore, as a peptide preparation, it possesses superior properties compared to traditional full-length proteins, facilitating subsequent clinical application and promotion.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides an SFRP2 polypeptide having:

[0011] (1) An amino acid sequence as shown in SEQ ID No. 1 or 2; or

[0012] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0013] (III) An amino acid sequence that has at least 80% sequence homology with the amino acid sequence shown in (I).

[0014] In some specific embodiments of the present invention, the SFRP2 polypeptide has:

[0015] (1) An amino acid sequence as shown in SEQ ID No. 2; or

[0016] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0017] (III) An amino acid sequence that has at least 80% sequence homology with the amino acid sequence shown in (I).

[0018] This invention also provides the use of the SFRP2 polypeptide in any of the following:

[0019] (1) Regulating osteogenic and / or odontoblast differentiation of mesenchymal stem cells; and / or

[0020] (2) Promote tissue repair and regeneration and / or regulate immune balance.

[0021] In some specific embodiments of the present invention, the mesenchymal stem cells include, but are not limited to, periapical papilla stem cells.

[0022] In some specific embodiments of the present invention, the tissue includes periodontal tissue.

[0023] In some specific embodiments of the present invention, the SFRP2 polypeptide is used in the preparation of medicaments for the prevention and / or treatment of periodontitis.

[0024] In some specific embodiments of the present invention, the prevention and / or treatment of periodontitis includes reducing CD4 levels. + The proportion of Th17 subsets in T cells and / or increased CD4+ + The proportion of Treg subsets in T cells;

[0025] The prevention and / or treatment of periodontitis also includes inhibiting CD4. + T cell differentiation into the Th17 subset and / or promotion of CD4+ differentiation + T cells differentiate into Treg subsets.

[0026] In some specific embodiments of the present invention, the prevention and / or treatment of periodontitis further includes:

[0027] (I) Promote the migration ability of DPSCs;

[0028] (II) Promotes the chemotactic ability of DPSCs;

[0029] (III) Promotes the in vitro odontogenic differentiation ability of DPSCs;

[0030] (IV) Rescuing the odontogenic differentiation ability of SFRP2shDPSCs;

[0031] (V) Promotes DPSCs-mediated intra-jawbone tooth regeneration capacity.

[0032] (VI) Inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0033] Based on the above research, the present invention also provides the application of the SFRP2 polypeptide in the preparation of medicaments for the prevention and / or treatment of colitis.

[0034] In some specific embodiments of the present invention, the prevention and / or treatment of colitis includes inhibiting CD4. + Proliferation of the Th17 subset in T cells and / or promotion of CD4+ + Proliferation of Treg subsets in T cells.

[0035] In some specific embodiments of the present invention, the prevention and / or treatment of colitis includes inhibiting the expression of pro-inflammatory proteins and / or promoting the expression of anti-inflammatory proteins.

[0036] In some specific embodiments of the present invention, the pro-inflammatory proteins include IL-17A and IL-22; the anti-inflammatory proteins include IL-10 and TGF-β1.

[0037] In some specific embodiments of the present invention, the prevention and / or treatment of colitis includes regulating the intestinal flora ratio.

[0038] In some specific embodiments of the present invention, the regulation of the intestinal flora ratio includes reducing the ratio of Firmicutes and / or Bacteroidetes.

[0039] In some specific embodiments of the present invention, the regulation of the intestinal flora ratio includes increasing the proportion of Muribacterium mulophila and / or Donorum spp.

[0040] The present invention also provides a medicine comprising the SFRP2 polypeptide.

[0041] The present invention also provides a pharmaceutical combination, including the said pharmaceutical product and any other active ingredients.

[0042] This invention provides SFRP2 functional peptides and their applications. The SFRP2 peptides of this invention have high purity; shorten the production cycle; allow for amino acid modification at both ends of the peptide to improve solubility; and produce amino acids as metabolites, making the products non-toxic. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0044] Figure 1 The bar chart showing the grayscale values ​​of the polypeptide chip in Example 1 is shown.

[0045] Figure 2 The results of the peptide chip array are shown in Example 1;

[0046] Figure 3 The purity, molecular weight, and solubility information of the control peptide ConPP and the functional peptide SFRP2-PP96 in Example 1 are shown.

[0047] Figure 4 The in vitro osteogenic induction results of different polypeptide groups in the experimental cases are shown.

[0048] Figure 5 The results of the in vitro migration ability of SFRP2 functional peptides on human dental pulp stem cells in the experimental cases are shown.

[0049] Figure 6 The results of the in vitro chemotactic activity of SFRP2 functional peptides on human dental pulp stem cells in the experimental cases are shown.

[0050] Figure 7 The results of the detection of Th17 and Treg subsets by the SFRP2 functional peptide in Example 2 are shown.

[0051] Figure 8 The results of detecting Th17 and Treg subsets by the SFRP2 functional peptide in Example 3 under Th17 polarization conditions are shown.

[0052] Figure 9 The results of detecting Th17 and Treg subsets by the SFRP2 functional peptide in Example 3 under Treg polarization conditions are shown.

[0053] Figure 10 This illustrates the therapeutic results of the SFRP2 functional peptide in Example 4 on experimental colitis in mice;

[0054] Figure 11 The results of the detection of Th17 and Treg subsets in mesenteric lymph nodes and colon tissue by the SFRP2 functional peptide in Example 5 are shown.

[0055] Figure 12Example 6 illustrates the detection of inflammation-related proteins in colon tissue using SFRP2 functional peptides;

[0056] Figure 13 This illustrates the effect of SFRP2 functional peptides on the regulation of gut microbiota in Example 7;

[0057] Figure 14 3D stereoscopic imaging of the therapeutic effect of SFRP2 functional peptide on experimental periodontitis in mice in Example 8;

[0058] Figure 15 The results of the in vitro odontogenic differentiation ability of SFRP2 functional peptides on human dental pulp stem cells are shown in Example 9;

[0059] Figure 16 In Example 9, the SFRP2 functional polypeptide partially rescued the in vitro odontogenic differentiation ability of SFRP2sh dental pulp stem cells.

[0060] Figure 17 This illustrates the method for establishing a rabbit jawbone intraosseous tooth regeneration model in Example 10;

[0061] Figure 18 This demonstrates the ability of SFRP2 functional peptides in Example 10 to promote tooth regeneration in rabbit jawbone mediated by dental pulp stem cells.

[0062] Figure 19 In Example 11, the SFRP2 functional peptide inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a. Detailed Implementation

[0063] This invention discloses SFRP2 functional peptides and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0064] The chemical synthesis technology of peptides is now quite mature, with relatively low research and development and synthesis costs. Furthermore, peptide products are easily separated from impurities, resulting in high purity. In addition, peptides can be easily modified by introducing non-natural amino acids during the production process. Since peptides are primarily degraded into amino acids through protein hydrolysis and eliminated through kidney filtration, their metabolites are non-toxic.

[0065] The SFRP2 peptide of this invention has high purity; shortens the production cycle; allows for amino acid modification at both ends of the peptide to improve solubility; and produces amino acids as metabolites, which are non-toxic.

[0066] After initially clarifying the regulatory role of the full-length SFRP2 protein in the Th17 / Treg balance and its therapeutic effect on periodontitis, we discovered through website searches and literature reviews that the SFRP2 protein contains an Fz region that can bind to the Wnt ligand. Finding the binding fragment between SFRP2 and the Wnt ligand for more precise use in subsequent research and clinical translation is a crucial challenge. Using peptide microarray analysis, we reported for the first time the key interaction sites between SFRP2 and the Wnt ligand. The peptide microarray results showed two sites with strong binding: sites 7-8 (named SFRP2-PP95, amino acid sequence: LQLCHGIEYQNMRLPNL, SEQ ID No. 1) and sites 49-54 (named SFRP2-PP96, amino acid sequence: AFGFPWPDMLECDRFPQDNDLCIPL, SEQ ID No. 2). Subsequently, we synthesized these two peptides and used ConPP (amino acid sequence: EEEACDQQPQEEEEKDEEGE, SEQ ID No. 3) as a control. The SFRP2 functional peptide can block the Wnt signaling pathway by binding to the putative Wnt ligand of the Frizzled receptor. To screen the optimal concentration of the functional peptide for cell action, based on the results of in vitro osteogenic differentiation experiments on human dental pulp stem cells (DPSCs), the peptide at site 49-54 (i.e., SFRP2-PP96) was selected for subsequent experimental studies.

[0067] SFRP2 functional peptide (SFRP2-PP96) can achieve the same immunomodulatory and periodontitis-promoting effects as recombinant SFRP2 protein. However, as a peptide preparation, it has superior properties compared to traditional full-length proteins, making it easier for subsequent clinical application and promotion.

[0068] The SFRP2 functional peptides provided by this invention and the raw materials and reagents used in their applications are all commercially available.

[0069] The present invention will be further illustrated below with reference to the embodiments:

[0070] Preparation example: Synthesis of peptide array chips:

[0071] 1. Synthesis of peptide array chips:

[0072] Based on the sequence of the SFRP2 protein (serial number Q96HF1), a peptide array was synthesized. Following an overlapping design, two peptide array chips were fabricated according to the standard operating procedure (SOP).

[0073] (1) The activated matrix chip membrane was placed on a fully automated peptide chip synthesizer (Aurora Group Company, VERSA110), and Fmoc-amino acid solution (Chengdu Chengnuo Biotechnology Co., Ltd., 20 kinds) was automatically transferred to a specific position on the activated membrane according to the program to react with the membrane;

[0074] (2) After each layer is synthesized, the membrane is sequentially immersed in blocking solution I (a DMF solution containing 2% (v / v) acetic anhydride (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10000317) of N,N-Dimethylformamide, anhydrous, amine free, 99.9%, alfa-asia catalog number: 043465) and blocking solution II (a DMF solution containing 2% (v / v) acetic anhydride and 2% (v / v) DIPEA (N,N-Diisopropylethylamine, Aldrich catalog number: 550043)) to block the side chains. Then, the membrane is washed 5 times with DMF (N,N-dimethylformamide, Sinopharm Chemical Reagent Co., Ltd., catalog number: CST12767S) for 3 minutes each time.

[0075] (3) The membrane was placed in a deprotection solution (DMF solution containing 20% ​​(v / v) piperidine (Sinopharm Chemical Reagent Co., Ltd., catalog number: 80104216) to remove the Fmoc- protecting group at the amino terminus. After deprotection, the membrane was washed 5 times with DMF for 3 minutes each time, and then washed 5 times with ethanol (Sinopharm Chemical Reagent Co., Ltd., catalog number: 100092008) for 3 minutes each time. The membrane was then dried. The above steps were repeated until the entire peptide array was synthesized.

[0076] (4) After the entire chip is synthesized, remove the side-chain protecting groups with piperidine, and wash the membrane 5 times with DMF for 3 minutes each time. Then wash the membrane 5 times with CH2Cl2 (dichloromethane, Sinopharm Chemical Reagent Co., Ltd., catalog number: 8004731916) for 3 minutes each time to remove all protecting groups on the peptides stepwise. Finally, wash the membrane with CH2Cl2, wash it 5 times with DMF for 3 minutes each time, and finally wash it with ethanol, wash it 5 times with DMF for 3 minutes each time, and let it air dry. The completed peptide array chip can be used immediately or stored at -20℃ / -80℃.

[0077] Example 1: Peptide Array Chip and Serum Immunoreaction Detection

[0078] (1) Peptide array chip sealing:

[0079] After activating the peptide array chip, add it to the blocking solution and shake it at 20℃±5℃ for 4 hours.

[0080] (2) Incubation of reactive protein samples with peptide array chips: The tested reactive protein Wnt3a was diluted with blocking buffer and co-incubated with the peptide array chip. The experimental group used 10 mL of 5 μg / mL Wnt3a recombinant protein reaction solution, while the control group used blocking buffer. The chips were shaken overnight at 4°C. The membranes were then removed and washed 5 times with TBST (Tris Buffered Saline with Tween 20, Tris salt buffer containing Tween) for 10 minutes each time.

[0081] (3) Incubation of peptide array chip with primary antibody: After diluting the primary antibody (Anti-Wnt3a antibody) at 1:1000, 10 mL of each of the experimental group and the control group were incubated at 20℃±5℃ for 2 hours, and the membrane was washed 5 times for 10 minutes each time.

[0082] (4) Incubation of peptide array chip with HRP-labeled secondary antibody: After diluting HRP-labeled secondary antibody at 1:10000, 10 mL of the experimental group and control group were incubated at 20℃±5℃ for 2 hours with shaking. The membrane was washed 5 times for 10 minutes each time.

[0083] (5) Color development: Add ECL luminescent reagent (Pierce ECL; Thermo Fisher Scientific; #32109, USA), Chempchemi digital imager (Vilber, France, VILBERFUSION FX7 Spectra), digital imaging.

[0084] Example of effect 1

[0085] 1. Synthesis of polypeptides

[0086] Based on the array results, sequences corresponding to obvious chromogenic spots were selected for peptide synthesis and subsequent functional verification.

[0087] 2. The array results are shown in Table 1. Figure 1 and Figure 2 .

[0088] Table 1

[0089]

[0090]

[0091] from Figure 1 , 2 The data can be analyzed to identify 7-8 points on the polypeptide array (amino acid sequences are:

[0092] LQLCHGIEYQNMRLPNL, SEQ ID No. 1) and points 49-54 (amino acid sequence is:

[0093] AFGFPWPDMLECDRFPQDNDLCIPL (SEQ ID No. 2) exhibits a colorimetric effect.

[0094] Subsequent in vivo and in vitro experiments were conducted using a peptide concentration of 10 μg / mL.

[0095] 3. The purity, molecular weight, and solubility information of the control peptide ConPP and the functional peptide SFRP2-PP96 are shown in [reference needed]. Figure 3 The control peptides ConPP and SFRP2-PP96 are both peptides produced in this invention.

[0096] As shown in the figure, the control peptide ConPP has a purity of 98.5% and a molecular weight of 2380.31, and can be dissolved in 100% H2O at a concentration of 1 mg / mL.

[0097] The functional polypeptide SFRP2-PP96 has a purity of 98.2% and a molecular weight of 2940.37. It can be dissolved in 100% DMSO at a concentration of 1 mg / mL.

[0098] Experimental example regarding the selection of SFRP2-PP95 and SFRP2-PP96

[0099] In vitro osteogenic induction was performed using fourth-generation (P4) DPSCs: cells were digested with trypsin (Gibco), centrifuged, resuspended in culture medium containing fetal bovine serum (FBS, Gibco), and counted using a hemocytometer to achieve a cell density of 1 × 10⁶ cells in 6-well plates. 5 Cells per well. When the cell density reached 70%, the culture medium was aspirated, and the cells were washed with phosphate-buffered saline (PBS, Biosharp) and replaced with osteogenic induction medium, which was changed every 2 to 3 days. The DPSCs group served as the control group. In the DPSCs+ConPP group, 10 μL / mL ConPP was maintained in the culture medium during osteogenic induction. In the DPSCs+SFRP2-PP95 group, 10 μL / mL SFRP2-PP95 was maintained in the culture medium during osteogenic induction. In the DPSCs+SFRP2-PP96 group, 10 μL / mL SFRP2-PP96 was maintained in the culture medium during osteogenic induction. After 14 days of continuous induction, the culture medium was discarded, and the cells were washed three times with PBS. Cells were fixed in tissue fixative (Beyotime) at room temperature for 30 minutes, washed three times with PBS, and 1 mL of 2% Alizarin Red solution (Sigma) was added to each well. After staining for 30 minutes, the staining solution was aspirated, and the cells were rinsed three times with PBS. The presence of mineralized nodules was observed under an inverted microscope, and images were acquired.

[0100] Add 10% CPC aqueous solution (hexadecylpyridine chloride, self-prepared) to each well to dissolve Ca2+ ions, 1 mL per well, and incubate at 20℃±5℃ for 30 minutes. Dilute the solution 1:10 and measure the absorbance (OD value) at 562 nm using a microplate reader. Statistical analysis software SPSS 22.0 and Prism 7.0 were used to process the experimental data. One-way ANOVA was performed to analyze the data. Data are expressed as mean ± SEM. P < 0.05 was considered statistically significant.

[0101] In vitro osteogenic induction was performed using fourth-generation (P4) DPSCs: cells were digested with trypsin (Gibco), centrifuged, resuspended in culture medium containing fetal bovine serum (FBS, Gibco), and counted using a hemocytometer to achieve a cell density of 1 × 10⁶ cells in 6-well plates. 5 Cells / well. When the cell density reaches 70%, the culture medium is aspirated, and the cells are washed with phosphate-buffered saline (PBS, Biosharp) and replaced with osteogenic induction medium. The osteogenic induction medium is changed every 2 to 3 days. Grouping is the same as above. After 7 consecutive days of induction, the culture medium is discarded, the cells are washed three times with PBS, and 600 μL of lysis buffer (Beyotime) is added. The cells are incubated at 37°C for 15 minutes. Cells are scraped and transferred to 1.5 mL EP tubes and centrifuged at 14000 rpm at 4°C for 10 minutes. 50 μL of Alkaline buffer sol, 50 μL of Stock Substrate sol, and 10 μL of lysed cell sample are added to each well of a 96-well plate and mixed well. The plates are incubated at 37°C for 15 minutes. The absorbance of the plates is measured at 405 nm.

[0102] The results showed that the DPSCs+SFRP2-PP95 and DFSCs+SFRP2-PP96 groups exhibited stronger mineralization capacity than the control group and the control peptide group. Figure 4 As shown in A). Quantitative detection results of calcium ions showed that the calcium ion concentrations in the DPSCs+SFRP2-PP95 and DFSCs+SFRP2-PP96 groups were significantly higher than those in the control group and the control peptide group, with SFRP2-PP96 showing a more significant effect than SFRP2-PP95. Figure 4 (See Table 2, B). ALP activity results showed that the SFRP2-PP96 group exhibited higher ALP activity, while the SFRP2-PP95 group did not show a statistically significant difference compared to the control group and the control peptide group. Figure 4 As shown in C (Table 2).

[0103] Table 2

[0104]

[0105] Cell scratch assay: DPSCs were scratched at 5 × 10⁻⁶.4 Seeds were planted at a density of 10 cells / well in 6-well plates and allowed to grow until 90% confluence. After serum-free culture for 24 hours, cross-scratches were made along the well diameter using a 10 μL pipette tip, followed by the addition of fresh culture medium. The scratches were observed under a microscope at 0h, 24h, and 48h from the same perspective to assess the degree of healing. ImageJ 1.49v software was used to detect blank areas, and the degree of healing was determined based on the percentage of blank areas in each group.

[0106] Transwell assay: Prepare a 0.1% collagen solution, according to 6–10 μg / cm³. 2 The cells were coated with a specific concentration of BSA onto Transwell chambers and incubated at 37°C for several hours. Excess liquid was removed from the coating surface, and the chambers were dried overnight. Serum-free culture medium containing BSA was added, and the chambers were incubated at 37°C for 30 minutes. Residual liquid in the culture plate was then aspirated. The cell density was adjusted to 2 × 10⁻⁶ cells / mL. 5 100 μL of DPSC cell suspension (24 h serum-free) was added to the upper chamber of a Transwell plate. The DPSCs had been cultured for 24 h without serum before preparing the cell suspension. 600 μL of α-MEM medium containing 5% fetal bovine serum was added to the lower chamber of a 24-well plate, and the plates were cultured for 6 h as usual. The Transwell chambers were then removed, the culture medium in the wells was discarded, and the cells were washed twice with calcium-free PBS. The plates were fixed with methanol for 30 min and then air-dried appropriately. The plates were stained with 0.1% crystal violet for 20 min, and the unmigrated cells on the upper layer were gently wiped away with a cotton swab. The plates were washed three times with PBS. Cells were observed and counted in five random fields of view under a 400x microscope.

[0107] The results showed that at 24, 48, and 72 hours, the DPSCs+SFRP2-PP95 group exhibited stronger migration ability than the control group and the control peptide group. Figure 5 As shown in Table 3 (A, B, C, and D); at 24 and 72 h, the DPSCs+SFRP2-PP96 group showed stronger migration ability than the control group and the control peptide group ( Figure 5 (A, B, and D are shown). Furthermore, at 24 and 48 hours, the DPSCs+SFRP2-PP95 group showed stronger chemotactic activity compared to the control group and the control peptide group. Figure 6 As shown in Table 4, A, B, and C; at 24 h, the DPSCs+SFRP2-PP96 group showed stronger chemotactic activity than the control group and the control peptide group. Figure 6 (A and B shown).

[0108] Table 3

[0109]

[0110] Table 4

[0111]

[0112] The results above indicate that the SFRP2-PP96 peptide may have stronger biological functions, such as promoting osteogenic differentiation of DPSCs. Therefore, SFRP2-PP96 was selected as the functional peptide of SFRP2 in subsequent experiments.

[0113] Example 2: SFRP2-PP96 reduces the proportion of Th17 subset and increases the proportion of Treg subset in CD4+ T cells.

[0114] To investigate the effects of SFRP2 functional peptides on CD4+ T cell subsets, especially Th17 and Treg subsets, flow cytometry and real-time RT-PCR were used for detection.

[0115] First, CD4 cells from C57BL / 6J mice were extracted, sorted, and activated in vitro. + T cells, in CD4 + T cell culture was treated with 10 μg / mL ConPP (control peptide) and SFRP2-PP96 for 3 days. Cells were then labeled with CD4 and stained with IL-17A intracellularly, and flow cytometry was used to detect Th17 subset staining. Alternatively, cells were labeled with CD4 and CD25 and stained with Foxp3 nuclear transcription factor, and flow cytometry was used to detect Treg subset staining. After flow cytometry analysis, lymphocyte gating, removal of adherent cells, and removal of dead cells were performed sequentially using Flowjo 10.4.0 software before CD4 analysis. + IL-17A + Th17 subgroup ratio and CD4 + CD25 + FoxP3 + The proportion of Treg subsets was observed. Results showed that, compared to the blank control group, CD4+ after ConPP treatment... + The proportions of Th17 and Treg subsets in T cells did not change significantly (P > 0.05). Figure 7 As shown in AB and DE), and CD4 after SFRP2-PP96 processing. + The proportion of Th17 subset in T cells was decreased (P < 0.05). Figure 7 As shown in Table 5 (AB), the proportion of the Treg subgroup increased (P < 0.05). Figure 7 DE is shown in Table 5).

[0116] Simultaneously, total RNA was extracted from the remaining replicates of the same batch of cells, and the relative mRNA expression levels of the key Th17 transcription factor RORγt and the key Treg transcription factor Foxp3 were detected using Real-time RT-PCR. The results showed that, compared to the blank control group, CD4+ expression levels after ConPP treatment were significantly higher. + The relative expression levels of RORγt and Foxp3 mRNA in T cells showed no significant changes (P > 0.05). Figure 7 The C and F shown are shown, while CD4 after SFRP2-PP96 processing is shown. + The relative expression level of RORγt mRNA in T cells was significantly decreased (P < 0.01). Figure 7 As shown in C (Table 6), the relative expression level of Foxp3 mRNA was significantly increased (P < 0.01). Figure 7 As shown in F (Table 6).

[0117] The above results suggest that, under in vitro conditions, the SFRP2 functional peptide SFRP2-PP96 reduces CD4 levels. + The peptide increased the proportion of the Th17 subset in T cells and increased the proportion of the Treg subset, while the control peptide had no such effect.

[0118] Table 5

[0119]

[0120] Table 6

[0121]

[0122] Example 3: SFRP2-PP96 inhibits the differentiation of CD4+ T cells into the Th17 subset and promotes their differentiation into the Treg subset.

[0123] Similarly, CD4 from C57BL / 6J mice was extracted, sorted, and activated in vitro. + T cells, in CD4 +T cell culture environments were inducing CD4+ T cells to differentiate into Th17 and Treg subsets by adding either 2 ng / mL TGF-β + 50 ng / mL IL-6 cytokine or 10 ng / mL TGF-β + 10 ng / mL IL-2 cytokine, respectively. Simultaneously, 10 μg / mL ConPP was added to one-third of the wells, and 10 μg / mL SFRP2-PP96 was added to the other one-third of the wells for 3 days. Cells were then subjected to CD4 surface labeling and IL-17A intracellular staining, and Th17 subset staining was detected by flow cytometry; alternatively, cells were stained with CD4 and CD25 surface labels and Foxp3 nuclear transcription factor, and Treg subset staining was detected by flow cytometry. After flow cytometry analysis, lymphocyte gating, removal of adhering cells, and removal of dead cells were performed sequentially using Flowjo 10.4.0 software before CD4 analysis. + IL-17A + Th17 subgroup ratio and CD4 + CD25 + FoxP3 + Treg subset proportions. Results showed that, compared to uninduced CD4... + T cells showed an increased proportion of Th17 subsets after the addition of Th17-inducing cytokines, but a slightly decreased proportion of Th17 subsets after treatment with SFRP2-PP96. Figure 8 As shown in AB (Table 7); correspondingly, compared to uninduced CD4 + T cells showed an increased proportion of Treg subsets after the addition of Treg-inducing cytokines, and the proportion of Treg subsets further increased after treatment with SFRP2-PP96 (*P<0.05). Figure 9 (See Table 9). However, the proportions of the Th17 and Treg subgroups did not change significantly after ConPP treatment.

[0124] Meanwhile, total RNA was extracted from the remaining replicates of the same batch of cells, and CD4+ was detected using Real-time RT-PCR. + Changes in the proliferation marker Ki-67 and the relative mRNA expression levels of RORγt and Foxp3 during T cell induction differentiation. Results showed that compared to uninduced CD4... + In T cells, the relative expression level of RORγt mRNA was significantly increased after the addition of Th17-inducing cytokines, while SFRP2-PP96 treatment inhibited the relative expression level of RORγt mRNA (***P<0.005). Figure 8 As shown in C (Table 8); however, after the addition of Treg-induced cytokines, the relative expression level of Foxp3 mRNA significantly increased (***P<0.005, Figure 9As shown in C (Table 10). The relative expression of RORγt and Foxp3 mRNA did not change significantly after ConPP treatment.

[0125] Table 7

[0126]

[0127] Table 8

[0128]

[0129] Table 9

[0130]

[0131] Table 10

[0132]

[0133] Example 4: SFRP2-PP96 inhibits experimental colitis in mice

[0134] Twenty-four 6-8 week old male C57BL / 6J mice were randomly divided into four groups: a normal control group, a 3% control group, and a 4% control group.

[0135] Six mice were assigned to each of the following groups: DSS+PBS, 3% DSS+ConPP, and DSS+SFRP2-PP96. Daily body weight, fecal viscosity, and fecal blood loss were recorded starting from the date of DSS administration in drinking water. Compared to the normal control group, DSS-induced experimental colitis mice showed significant weight loss and dull fur from day 6, and significant fecal blood loss and diarrhea from day 4, which progressively worsened.

[0136] The change in body weight of mice on the day they began drinking DSS water (day 0) was considered as 1. The daily body weight of the mice was recorded. After the experiment, the percentage change and rate of decrease in daily body weight compared to day 0 were calculated, and line graphs showing the body weight changes of the four groups of mice were plotted. Figure 10 As shown in Table 11 (A), the line graph shows that from day 6, the body weight of mice in the DSS+PBS and DSS+ConPP groups decreased significantly until the end of the experiment (P < 0.01); the body weight change of mice in the DSS+SFRP2-PP96 group was not statistically different from that of the normal control group (P > 0.05), but was significantly higher than that of the DSS+PBS and DSS+ConPP groups (P < 0.005). The results indicate that intraperitoneal injection of SFRP2 functional peptides can inhibit the trend of body weight loss in mice with experimental colitis.

[0137] Using the normal control group as a reference (considered as 0), the disease activity indices calculated from the four groups of mice were plotted as a line graph. Figure 10As shown in B (Table 12). The line graph shows that from day 4 onwards, the DAI of mice in the DSS+PBS group and the DSS+ConPP group was significantly higher than that in the normal control group and the SFRP2 functional peptide treatment group (P < 0.005), while there was no statistically significant difference in DAI between the SFRP2 functional peptide group and the normal control group (P > 0.05).

[0138] Mice were sacrificed on the seventh day after the start of DSS (Diverterless Supersedation) drinking water therapy. The colon, mesenteric lymph nodes, serum, and feces were dissected and collected. Head-down images of the colons of the four groups of mice were taken using a ruler. Figure 10 As shown in Figure C), the colon of mice with DSS-induced experimental colitis was significantly shortened. The colon length in the ConPP treatment group was close to that in the DSS+PBS group, while the colon in the SFRP2 functional peptide treatment group was longer than that in the DSS+PBS group, but still slightly shorter than that in the normal control group. Statistical analysis of the colon length of the four groups of mice was performed and a bar chart was plotted. Figure 10 As shown in D (Table 13), the results showed that the colon length of mice in the DSS+PBS group and the DSS+ConPP group was significantly lower than that in the normal control group (P<0.01, P<0.005) and the DSS+SFRP2-PP96 group (P<0.01, P<0.005), while there was no statistically significant difference between the normal control group and the DSS+SFRP2-PP96 group (P>0.05).

[0139] Next, the colon tissues of the four groups of mice were subjected to pathological H&E staining ( Figure 10 As shown in E), the results showed that the colonic epithelial structure of the normal control group mice was intact, and no inflammatory cell infiltration was observed. Compared with the normal control group, the DSS-induced experimental colitis group showed partial mucosal or muscular layer defects, goblet cell loss, and infiltration of inflammatory cells such as lymphocytes and neutrophils. No significant reduction in inflammatory infiltration was observed in the DSS+ConPP group mice. In contrast, the degree of damage to the colonic mucosa of the SFRP2-PP96 treatment group mice was reduced, the mucosa or muscular layer was basically intact, the number of lost goblet cells was reduced, and the inflammatory cell infiltration was significantly reduced.

[0140] Histological pathological scoring was performed on the above H&E stained sections. Figure 10 As shown in Table 13), the colon histopathological score of mice in the DSS+PBS group was significantly higher than that of the normal control group (P<0.005), while the colon histopathological score of mice in the DSS+recombinant SFRP2 protein group was reduced (P<0.01).

[0141] Table 11

[0142]

[0143]

[0144] Table 12

[0145]

[0146] Table 13

[0147]

[0148]

[0149] Example 5: SFRP2-PP96 inhibits Th17 proliferation and promotes Treg proliferation in colon tissue.

[0150] Mesenteric lymph nodes were collected from mice in each group. After treatment with a cell stimulant (including a protein transport inhibitor, Invitrogen) for 5 hours, CD4 surface labeling and IL-17A intracellular staining were performed; alternatively, cells were directly labeled with CD4 and CD25 surface markers and stained with Foxp3 nuclear transcription factors. Flow cytometry analysis was performed, followed by lymphocyte gating, removal of adherent cells, and removal of dead cells using Flowjo 10.4.0 software. The proportions of the CD4+IL-17A+Th17 subset and the CD4+CD25+Foxp3+Treg subset were then analyzed. Results showed that compared with the normal control group, the proportion of Th17 subset among CD4+ T cells in the mesenteric lymph nodes of DSS-induced colitis mice was increased (P < 0.01), while the proportion of Th17 subset decreased after treatment with the SFRP2 functional peptide SFRP2-PP96 (P < 0.01), with statistically significant differences. Figure 11 (See Table 14 for A and B). Furthermore, compared to the normal control group, the proportion of Treg subsets among CD4+ T cells in the mesenteric lymph nodes of DSS-induced colitis mice was decreased (P < 0.05), while the proportion of Treg subsets increased after treatment with the SFRP2 functional peptide SFRP2-PP96 (P < 0.05), and the differences were statistically significant. Figure 11 The CD values ​​are shown in Table 14. ConPP treatment had no significant effect on the proportions of the Th17 and Treg subsets (P > 0.05).

[0151] To further investigate the effects of SFRP2 functional peptides on the number of Th17 and Treg cells in colon tissue, immunohistochemical staining of RORγt and FOXP3 was performed on paraffin sections of colon tissue from mice in each group. The results showed that the number of RORγt-positive cells in the colon tissue of the normal control group was low, while the number of RORγt-positive cells in the DSS+PBS and DSS+ConPP groups was significantly increased (P < 0.005). In contrast, the number of RORγt-positive cells in the colon tissue of the DSS+SFRP2-PP96 treatment group was similar to that of the normal control group and significantly lower than that of the DSS+PBS and DSS+ConPP groups (P < 0.01). Figure 11 (See Table 15 for E and F). Furthermore, the number of FOXP3-positive cells in the colon tissue of the normal control group was low, while the number of FOXP3-positive cells in the colon tissue of the DSS+PBS and DSS+ConPP groups was slightly increased. However, the number of FOXP3-positive cells in the colon tissue of the DSS+SFRP2-PP96 group was significantly increased (P < 0.01). Figure 11 E and G are shown in Table 15.

[0152] Table 14

[0153]

[0154] Table 15

[0155]

[0156] Example 6: SFRP2-PP96 inhibits the expression of pro-inflammatory proteins and promotes the expression of anti-inflammatory proteins in inflamed colon tissue.

[0157] A segment of colon tissue was collected from each group of mice for inflammation-related protein microarray analysis. The expression levels of 18 proteins were detected.

[0158] Further quantitative analysis revealed significant differences in the expression levels of four proteins in the colon tissues of different groups: IL-17A, IL-22, IL-10, and TGF-β1. Bar chart results showed that the expression levels of pro-inflammatory proteins IL-17A and IL-22 in the colon tissues of colitis mice were significantly higher than those in the normal control group (P < 0.05). Figure 12 As shown in Table 16 (A and B), after ConPP treatment, the expression levels of IL-17A and IL-22 did not decrease significantly (P > 0.05). Figure 12 As shown in Table 16, A and B, after treatment with SFRP2-PP96, showed a significant decrease in their expression levels (P < 0.05). Figure 12(See Table 16 for A and B). Furthermore, the expression levels of anti-inflammatory proteins IL-10 and TGF-β1 in the colon tissue of colitis mice were significantly lower than those in the normal control group (P < 0.05, P < 0.005). Figure 12 As shown in Table 16 (C and D), treatment with the control peptide ConPP had no significant effect on its expression level (P > 0.05). Figure 12 As shown in C and D (Table 16), after treatment with the SFRP2 functional peptide SFRP2-PP96, the expression levels of IL-10 and TGF-β1 were significantly increased (P < 0.05, P < 0.01). Figure 12 C and D are shown in Table 16.

[0159] Table 16

[0160]

[0161] Example 7: SFRP2-PP96 may promote colitis recovery by regulating gut microbiota ratio.

[0162] 16S rDNA sequencing analysis was performed on the intestinal contents of each group of mice, and the data were integrated. We also performed phylum and genus-level analysis of the fecal microbiota of each group of mice. Figure 13 As shown in Figure A, compared with the normal control group, the number of Firmicutes (orange) and Bacteroidetes (blue) was decreased in both the DSS+PBS group and the DSS+ConPP group, and the Firmicutes / Bacteroidetes ratio was also decreased (p < 0.05). Figure 13 As shown in B (Table 17); in the DSS+SFRP2-PP96 group, compared with the normal control group, the number of Firmicutes (orange) and Bacteroidetes (blue) was reduced, but the Firmicutes / Bacteroidetes ratio increased to near the level of the normal control group, and was significantly higher than that of the DSS+PBS group and the DSS+ConPP group (P<0.01, P<0.05). Figure 13 As shown in B (Table 17), SFRP2 functional peptide treatment can restore some of the balance of gut microbiota.

[0163] At the genus level, the dominant bacteria in the normal control group was *Muribaculaceae*, while the relative abundance of *Muribaculaceae* in the DSS+PBS group was lower than that in the normal control group (P < 0.005). Figure 13 As shown in Tables 17 (C and D), the relative abundance of *Muribaculaceae* did not change significantly after ConPP treatment, while the relative abundance of *Muribaculaceae* increased significantly after SFRP2-PP96 treatment (P < 0.01). Figure 13(See Table 17, C and D). The variation trend of Lactobacillus (Lachnospiraceae) in the four groups of mice was similar to that of Muribaculaceae (P < 0.01). Figure 13 (See Table 17 for C and E). In the DSS+SFRP2-PP96 group, *Dubosiella* became the dominant bacterium, significantly higher than in the other two groups (P < 0.05, P < 0.005). Figure 13 C and F are shown in Table 17.

[0164] The above results demonstrate that SFRP2-PP96 helps improve gut microbiota imbalance and promotes recovery from enteritis.

[0165] Table 17

[0166]

[0167] Example 8: SFRP2-PP96 Treatment of Experimental Periodontitis in Mice

[0168] Three weeks after treatment with SFRP2 functional peptide injections, mice were sacrificed, and bilateral maxillae were harvested. RNA was extracted from palatal soft tissue, and the maxillary bone tissue was fixed with 4% paraformaldehyde before micro-CT imaging. The therapeutic effect on experimental periodontitis in mice was evaluated using imaging techniques.

[0169] 3D stereoscopic imaging results as follows Figure 14 The height from the cementoenamel junction to the alveolar ridge crest was measured, i.e., clinical attachment loss (AL, CEJ-AB). The results are shown in Table 18. Compared with the normal control group, the CEJ-AB of mice in the NaCl control group and the ConPP treatment group was significantly increased (P<0.01), while the CEJ-AB of the SFRP2-PP96 treatment group was restored (P<0.05).

[0170] Table 18

[0171]

[0172]

[0173] Example 9: SFRP2-PP96 promotes the odontogenic differentiation ability of DPSCs in vitro and can partially salvage the odontogenic differentiation ability of SFRP2sh DPSCs.

[0174] To investigate the effect of SFRP2 functional peptides on the odontogenic differentiation function of DPSCs in vitro, alkaline phosphatase (ALP) activity was measured, alizarin red staining was performed, calcium ion quantification was conducted, and Western blot experiments were conducted.

[0175] Tooth induction medium was prepared by adding 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mmol / L L-glutamine, 0.05 mmol / L ascorbic acid, 10 mmol / L sodium β-glycerophosphate, and 10 nmol / L dexamethasone to α-MEM culture medium. 10 μg / mL ConPP (control peptide) and SFRP2-PP96 were added to the medium. DPSCs were then cultured at 1×10⁻⁶... 5 The cells were seeded at a concentration of 1 cell per well in 6-well plates. After the cells grew to 70%, the medium was replaced with tooth-inducing medium, and the medium was changed every 3 days.

[0176] Seven days after tooth induction, the alkaline phosphatase activity of DPSCs was measured using an ALP activity kit (Sigma-Aldrich, USA). Optical density (OD) values ​​were measured using a microplate reader (absorbance 405 nm), and the ALP activity of the samples was calculated using a standard ALP activity curve. The results showed that ConPP had no significant effect on the ALP activity of DPSCs (P>0.05). Figure 17 A), while SFRP2-96 peptide significantly enhanced the ALP activity of DPSCs (P<0.01, Figure 15 A).

[0177] Two weeks after odontogenic induction culture, cells were fixed with tissue fixative (Beyond) for 1 hour and stained with 2% Alizarin Red (Sigma-Aldrich, USA). Calcium ion concentration was determined: Alizarin Red dye was destained by incubation at 20℃±5℃ for 30 minutes with 10% hexadecylpyridine chloride (dissolved in 10 mmol / L sodium phosphate), and the OD value was measured using a microplate reader (absorbance 562 nm). The calcium ion concentration of the samples was calculated using a standard calcium ion concentration curve. Results showed that, compared with the control group, ConPP had no significant effect on the Alizarin Red staining intensity and calcium ion concentration of DPSCs (P>0.05). Figure 15 B- Figure 15 C), while SFRP2-96 peptide significantly enhanced the Alizarin Red staining intensity and calcium ion concentration of DPSCs (P<0.01, Figure 15 B Figure 15 -C).

[0178] After 7 days of odontogenic induction culture, intracellular proteins were extracted, and the levels of intracellular DSPP and DMP1 proteins were detected by Western blot. The results showed that ConPP had no significant effect on the intracellular DSPP and DMP1 protein levels in DPSCs. Figure 15 D), while SFRP2-96 peptide significantly enhanced the intracellular DSPP and DMP1 protein content of DPSCs ( Figure 15 D).

[0179] To investigate the rescue effect of SFRP2 functional peptides on SFRP2 knockdown DPSCs, alkaline phosphatase (ALP) activity was measured, alizarin red staining was performed, and Western blot experiments were conducted.

[0180] Short hairpin RNAs (shRNAs) were complementary to the target gene SFRP2 and were subcloned into the pLKO.1 lentiviral vector (Addgene, USA). Scramble shRNAs (Scramsh) were used as a knockout control virus. The target sequence of SFRP2shRNAs is as follows: 5'-ttgatgtaggttatctccttc-3'. Scramble shRNAs and SFRP2shRNAs were transfected into DPSCs, and the knockdown efficiency of SFRP2 was detected by Real-time RT-PCR. The results showed that SFRP2 RNA expression was significantly reduced in the SFRP2sh group (P<0.01). Figure 16 A). Stable transfected DPSCs were subjected to odontogenic induction, and 10 μg / mL of SFRP2-PP96 was added to the SFRP2sh group. ALP activity was measured 5 days after odontogenic induction, and it was found that ALP activity in the SFRP2sh group was significantly lower than that in the Scramsh group (P<0.01). Figure 16 B), but it can be partially salvaged by SFRP2-PP96 (P<0.05, Figure 16 B). Similarly, alizarin red staining results after 2 weeks of tooth formation induction showed that the alizarin red staining depth in the SFRP2sh group was significantly lower than that in the Scramsh group. Figure 16 C), but it can be partially salvaged by SFRP2-PP96 ( Figure 16 C). Furthermore, 7-day results from tooth formation induction showed that intracellular DSPP and DMP1 protein expression decreased after SFRP2 knockout. Figure 16 D), but can be partially salvaged by SFRP2-PP96 ( Figure 16 D).

[0181] Example 10: SFRP2-PP96 promotes DPSCs-mediated intraosseous tooth regeneration.

[0182] To investigate the effect of SFRP2-PP96 on DPSC-mediated intraosseous tooth regeneration, the right maxillary and mandibular incisors of rabbits were extracted, and a mixture of 10 μg / ml ConPP or SFRP2-PP96 cells and a hydrogel scaffold was reimplanted and sutured to establish a rabbit intraosseous tooth regeneration model. Figure 17 ).

[0183] Three months later, the animals were euthanized, and microCT scans were performed to determine the volume of regenerated high-density calcification within the jawbone. Results showed that ConPP had no significant effect on the volume of regenerated high-density calcification within the jawbone compared to the control group (P>0.05). Figure 18 A, Figure 18 B), while the volume of regenerated density calcification in the jawbone was significantly increased in the SFRP2-PP96 treatment group (P<0.05). Figure 18 A, Figure 18 B). Gross observation of the jawbone section showed no significant difference between the ConPP group and the control group, while the SFRP2-96 peptide treatment group formed significantly more high-density calcifications than the control group. Figure 18 C).

[0184] Further histological staining of rabbit jawbone sections revealed no significant difference between the ConPP group and the control group in HE staining. However, the SFRP2-96 peptide-treated group showed the formation of numerous dentin-like regenerative organisms surrounded by a large number of odontoblast-like cells. Figure 18 D). Masson staining results also showed no significant difference between the ConPP group and the control group, but treatment with SFRP2-96 peptides resulted in the formation of more collagen and osteoid (D). Figure 18 E). Furthermore, both immunohistochemical and immunofluorescence staining results showed that the expression intensity of DSPP and DMP1 in the jawbone was significantly stronger in the SFRP2-96 peptide-treated group. Figure 18 F, Figure 18 G), there was no significant difference between the ConPP group and the control group. Figure 18 F, Figure 18 G).

[0185] Example 11: SFRP2-PP96 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0186] To investigate the binding affinity of SFRP2-PP96 to Wnt3a, microscale thermophoresis (MST) was used. Recombinant Wnt3a protein and SFRP2-PP96 were placed in a capillary tube at a specific concentration gradient. Infrared laser heating generated a microscopic temperature gradient field, causing thermophoresis and resulting in changes in the fluorescence distribution within the reaction system. The results showed a Kd value of 8.723E-6, indicating that SFRP2-PP96 can bind to Wnt3a in vitro. Figure 19 A).

[0187] The effect of SFRP2-PP96 on intracellular Wnt / β-catenin in DPSCs was further investigated. ConPP and SFRP2-PP96 were added to the culture medium, and intracellular proteins were collected after 24 h of treatment and verified by Western blot experiments. The results showed that among the three groups, total intracellular β-catenin levels were significantly different, but SFRP2-PP96 enhanced intracellular p-β-catenin levels and inhibited the Wnt / β-catenin signaling pathway. Figure 19 B), ConPP had no significant effect on intracellular p-β-catenin levels. Figure 19 B). Furthermore, intracellular p-β-catenin levels decreased after SFRP2 knockout, but increased after the addition of SFRP2-PP96. Figure 19 C).

[0188] Therefore, it can be concluded that SFRP2-PP96 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0189] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An SFRP2 polypeptide, characterized in that, The amino acid sequence of the SFRP2 polypeptide is shown in SEQ ID No.

2.

2. The use of the SFRP2 polypeptide as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of periodontitis.

3. The use of the SFRP2 polypeptide as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of colitis.

4. A drug, characterized in that, Includes the SFRP2 polypeptide as described in claim 1.

5. A drug combination, characterized in that, Includes the drug as described in claim 4 and any other active ingredients.

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