Dentin inducer for enhancing histone succinylation and application thereof

Abstract

The invention provides a dentin inducer for enhancing histone succinylation and application of the dentin inducer. The dentin inducer is dimethyl succinate. The invention finds that the Wnt3a protein with a definite dental differentiation effect promotes dental pulp stem cell dental differentiation after inducing histone succinic acid by increasing the content of succinic acid in cells, and the Wnt3a substitution effect can be played by directly adding succinic acid or succinic acid derivatives such as dimethyl succinate; the stem cell histone succinylation is enhanced; meanwhile, the dental pulp stem cell dental differentiation is promoted. Moreover, compared with a Wnt3a protein inducer, the succinic acid or the succinic acid derivative has the advantages of being stable in preparation and clear in target spot, is more suitable for clinical operation, and has important practical significance for promoting clinical transformation of a dental pulp and dentin complex regeneration technology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pulp-dentin complex regeneration, and particularly relates to a dentin inducer for enhancing histone succinylation and application thereof. BACKGROUND

[0002] As a newly discovered post-translational modification of histone, histone lysine succinylation (Ksuc) has attracted extensive attention from researchers in recent years. This modification changes the chemical environment of lysine residues by adding succinyl groups, thereby affecting the interaction between histone and DNA and having a profound impact on cell fate and function. Known studies have found that abnormal regulation of histone lysine succinylation plays an important role in the occurrence and development of cancer. In addition, studies have shown that histone lysine succinylation also plays an important role in the process of hepatitis B virus infection.

[0003] Strengthening the research on the interaction between histone lysine succinylation and cell metabolism will help reveal the regulatory network of cell physiological functions and provide a deeper theoretical basis for the development of life sciences. With the continuous deepening of research, it is believed that histone lysine succinylation modification will have greater application potential in the field of life sciences and health medicine.

[0004] In recent years, with the rapid development of stem cell biology and tissue engineering, stem cell-based pulp-dentin complex regeneration strategies have shown good application prospects. Among them, dental pulp stem cells (DPSCs) have become the focus of dental regenerative medicine research due to their wide source, easy access, and multi-directional differentiation potential.

[0005] Pulp-dentin complex regeneration has important clinical significance for maintaining tooth vitality, restoring tooth function, and prolonging tooth service life. Currently, it mainly relies on protein preparations such as Wnt3a and Wnt10a to induce dental pulp stem cells to differentiate and promote dentin regeneration. These growth factors activate specific signaling pathways to regulate the proliferation and differentiation of stem cells, thereby promoting dentin formation.

[0006] However, these protein molecules face many technical obstacles in the application process. In terms of mechanism: the molecular mechanism of these growth factors in promoting dentin regeneration is not fully understood, especially the mechanism of action at the level of cell metabolism regulation is still unclear, which also affects the optimization and standardization of treatment effect to some extent. In terms of application: first, the inherent instability of protein molecules leads to rapid degradation in vivo, extremely low bioavailability, and difficulty in maintaining a sustained and effective biological concentration. Second, there are significant limitations in the delivery and positioning of protein preparations, which cannot accurately control the target and concentration gradient, thereby affecting the induction efficiency. At the same time, the high cost of protein purification and preparation and the complex production process also seriously restrict its wide application.

[0007] Therefore, in order to overcome the limitations of existing protein preparations, improve treatment effect, and reduce treatment cost, the present application attempts to develop a new type of dentin inducer with good stability, high bioavailability, clear mechanism of action and good cost-effectiveness by using succinylation mechanism, which has important practical significance for promoting the clinical transformation of dental pulp dentin complex regeneration technology. SUMMARY

[0008] In view of the shortcomings of the prior art, the purpose of the present application is to provide a dentin inducer with enhanced histone succinylation and its application.

[0009] To achieve this purpose, the present application adopts the following technical solutions:

[0010] In a first aspect, the present application provides a dentin inducer with enhanced histone succinylation, which comprises succinic acid or a succinic acid derivative.

[0011] Wherein, the succinic acid derivative comprises succinic acid ester, succinic acid amide or a pharmaceutically acceptable salt thereof.

[0012] It is found in the present application that Wnt3a protein with a clear odontogenic differentiation effect promotes odontogenic differentiation of dental pulp stem cells by increasing intracellular succinic acid content to induce histone succinic acid. On this basis, using Wnt3a as a positive control, it is found through experiments that the use of succinic acid or succinic acid derivatives can have the same effect as Wnt3a, can significantly enhance the level of histone succinylation, and then promote the transcriptional activation of odontogenic differentiation related genes. That is, succinic acid or succinic acid derivatives, by increasing the concentration of succinic acid to promote succinylation, can be used as a new dentin inducer to replace Wnt3a to enhance the histone succinylation of stem cells and promote odontogenic differentiation of dental pulp stem cells. The succinylation refers to the process of covalently binding succinyl groups to amino acid residues (mainly lysine K) through enzymatic or non-enzymatic methods.

[0013] In the present application, the succinic acid or succinic acid derivative includes succinic acid, dimethyl succinate and other small molecule substances capable of providing succinyl group and increasing the level of succinylation.

[0014] As a preferred technical solution of the present application, the dentin inducer includes any one or a combination of at least two of succinic acid (Succ) and dimethyl succinate (E-Succ), and preferably dimethyl succinate.

[0015] In a second aspect, the present application provides a use of the dentin inducer according to the first aspect in the preparation of a tooth differentiation promoting product.

[0016] As a preferred technical solution of the present application, the tooth differentiation promoting product includes any one or a combination of at least two of a pharmaceutical preparation, a cell culture medium or an implant material.

[0017] In the present application, the tooth differentiation promoting product includes various forms of biological products. Among them, the succinic acid derivative such as dimethyl succinate can be used directly, i.e. directly placing dimethyl succinate in the tooth root canal, or adding the dimethyl succinate in the culture medium to treat stem cells in vitro, and then transplanting the stem cells into the tooth root canal. In addition, the dimethyl succinate can also be prepared into different types of implant materials with other drugs or excipients and placed in the tooth root canal. Since succinic acid can start other signal pathways in the body and cannot directly enter the cell, it is necessary to use other auxiliary materials or combine with other substances that can enter the cell to smoothly enter the cell and play a role in increasing the concentration of succinic acid. For example, the present application can prepare nanoparticles containing succinic acid or carry succinic acid through a scaffold to introduce it into the cell.

[0018] As a preferred technical solution of the present application, the concentration of the dentin inducer in the cell culture medium is 250 nM-1 μM; for example, it can be 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1 μM, etc. Dimethyl succinate can be directly added to the cell culture medium. If succinic acid is needed as a dentin inducer, it needs to be prepared into nanoparticles or other forms that can enter the cell.

[0019] As a preferred technical solution of the present application, the implant material contains hydrogel material and / or scaffold material; the hydrogel material or scaffold material is mainly used to carry succinic acid and introduce it into the cell.

[0020] In a third aspect, the present application also provides use of succinic acid or a succinic acid derivative in the preparation of a histone H3K14 succinylation inducer.

[0021] The present application takes dimethyl succinate as an example to verify the target point of succinic acid or a succinic acid derivative through experiments. Dimethyl succinate induces a large number of key odontogenic genes to be activated by improving histone succinylation, and induces odontogenic differentiation. More specifically, it induces stem cells to initiate odontogenic differentiation by enhancing the succinylation of the H3K14 site. Prior to this, there has been no related research to propose a theory of inducing odontogenic differentiation based on enhancing histone succinylation. The present application clearly identifies one of the key mechanisms and target points of inducing stem cell odontogenic differentiation.

[0022] In a fifth aspect, the present application also provides a dentin inducer for inhibiting the function of desuccinylase, which comprises a Sirt5 inhibitor and / or a Sirt7 inhibitor.

[0023] Since enhancing histone succinylation can induce stem cell odontogenic differentiation, small molecule preparations for inhibiting the function of desuccinylase in dental pulp stem cells can also enhance histone succinylation, including Sirt5 inhibitors (NRD167, etc.), Sirt7 inhibitors (SIRT7 inhibitor 97491, etc.), and other small molecule drugs.

[0024] The numerical range described in the present application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to the limited space and the consideration of simplicity, the present application does not list all the specific point values included in the range.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] 1. The present application first identifies that succinic acid or a succinic acid derivative can induce dental pulp stem cell odontogenic differentiation, and the induction effect is similar to that of the positive control Wnt3a, which is efficient and stable. In addition, the inorganic salt component has the advantages of stable preparation and clear target point compared with protein preparations, and is more suitable for clinical operation.

[0027] 2. The present application confirms that succinic acid or a succinic acid derivative induces a large number of key odontogenic genes to be activated by improving histone succinylation, and induces odontogenic differentiation. Specifically, it provides to enhance the succinylation of the H3K14 site, thereby inducing stem cells to initiate odontogenic differentiation. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 is a diagram of the H3K14su CUT&Tag experimental results after Wnt3a induction in Example 1.

[0029] Figure 2Figure 2 shows the results of alizarin red staining of the dental pulp stem cells in each experimental group and the control group in Example 2 after induction.

[0030] Figure 3 Figure 3 shows the comparison of the expression of proteins related to the increase of dental differentiation of the dental pulp stem cells in each experimental group and the control group in Example 2, wherein Figure I is a gel map of the protein expression; Figure II is a columnar chart of the relative expression of DSPP protein; and Figure III is a columnar chart of the relative expression of DMP1 protein.

[0031] Figure 4 Figure 4 shows the comparison of the level of succinylation of histone H3 and H2B in each experimental group and the control group in Example 2 after induction. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further illustrated below in conjunction with the drawings and through specific embodiments, but the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0033] In the following examples, unless otherwise specified, the reagents and consumables used are purchased from conventional reagent manufacturers in the field; and unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the field.

[0034] Example 1 Cell grouping culture

[0035] Example 1 Exploration of transcriptional activation sites of genes related to dental differentiation

[0036] At present, it is known that Wnt3a can induce dental differentiation, and in order to further explore its mechanism, this embodiment studies the activation sites related thereto through CUT&Tag experiment.

[0037] (1) Cell culture

[0038] The human dental pulp stem cells used in this embodiment are isolated from wisdom teeth of healthy donors aged 18 to 25 years old, and are cultured in accordance with the protocol approved by the Ethics Committee of Capital Medical University.

[0039] The dental pulp stem cells are cultured in α-MEM (Gibco) at 37°C and 5% CO2, and 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin and 100 mg / mL streptomycin (Gibco) are added.

[0040] To induce odontogenesis, cells were cultured in odontogenic differentiation medium (including complete growth medium, supplemented with 50 mg / L ascorbic acid, 10 mmol / L β-glycerophosphate and 100 nmol / L dexamethasone), and further supplemented with 50 ng / mL recombinant human Wnt3a (R&D Systems, 5036-WN-010).

[0041] (2) CUT&Tag experiment

[0042] Procell anti-mycoplasma reagent was used to completely eliminate mycoplasma contamination, and the decontamination effect was verified by Yeasen MycAway™ Plus-Color One-Step Detection Kit.

[0043] In the experimental process, cells were coupled with capreomycin A coated magnetic beads to realize cell fixation through surface glycoprotein mediation. Cell membrane permeability was achieved by treating with digital mycin, and then specific antibodies were introduced to achieve precise binding of target proteins.

[0044] Superactive pG-Tn5 / pA-Tn5 transposase was used to precisely locate Tn5 enzyme through Fc region protein G / A domain interaction. In the optimized buffer system, transposase simultaneously completed chromatin fragmentation and Illumina sequencing adapter ligation. DNA fragments were purified by phenol-chloroform extraction, and PCR amplification and concentration were performed using adapter-specific primers. Library quality was evaluated by Agilent 2100 Bioanalyzer and Qubit, followed by high-throughput sequencing on the Illumina NovaSeq platform.

[0045] Bioinformatics analysis includes Trimmomatic quality trimming, reference genome alignment (FastQC assesses data integrity), MACS2 peak identification (IDR filtering), ChIPseeker genome feature annotation, HOMER de novo motif analysis, and DiffBind differential peak analysis.

[0046] As shown in Figure 1 , using CUT&Tag experiment, it was found that the increase in H3K14 succinylation level was accompanied by significant enrichment of H3K14su signal in the gene promoter region, suggesting that induced dental pulp stem cells activated differentiation-related genes and accelerated their odontogenic differentiation process. That is, Wnt3a-induced odontogenic differentiation is related to its ability to enhance the level of H3K14 succinylation.

[0047] Example 2 Verification of the induction effect of succinylation derivatives

[0048] Example 1 shows that Wnt3a can enhance the level of H3K14 succinylation. Due to the limitation of protein preparation and the fact that succinic acid and its derivatives often induce protein succinylation, this example attempts to verify whether succinylated derivatives can enhance the level of H3K14 succinylation, and thus replace Wnt3a as a new dentin inducer.

[0049] The specific steps are as follows:

[0050] (1) The cell culture steps are the same as in Example 1.

[0051] (2) Grouping: control group, succinic acid group (Succ), and dimethyl succinate group (E-Succ).

[0052] The control group is induced by odontogenic differentiation medium, the succinic acid group is added with 1 μM succinic acid (Sigma) to the odontogenic differentiation medium, and the dimethyl succinate group (a total of five groups) is added with gradient concentrations of dimethyl succinate (Sigma) to the odontogenic differentiation medium. The specific concentrations are as follows:

[0053] Experimental group ① contains 250 nM of E-Succ;

[0054] Experimental group ② contains 500 nM of E-Succ;

[0055] Experimental group ③ contains 1 μM of E-Succ;

[0056] Experimental group ④ contains 2 μM of E-Succ;

[0057] Experimental group ⑤ contains 4 μM of E-Succ.

[0058] (3) Verification experiment

[0059] 1. Alizarin red S staining: To evaluate odontogenic differentiation, alizarin red S solution (Cyagen) was used to analyze calcium deposition at predetermined time points.

[0060] As shown in Figure 2 , the results of alizarin red staining showed that the addition of 250 nM-1 μM dimethyl succinate can enhance the formation of calcium nodules, and beyond this concentration, there is no obvious enhancement effect.

[0061] 2. Determination of odontogenic differentiation-related protein expression and histone succinylation sites

[0062] Total cell protein was extracted using RIPA lysis buffer (Beyotime) containing protease inhibitor cocktail (Beyotime) and 1 mM PMSF.

[0063] Histones were collected using a histone extraction kit (abcam).

[0064] Protein samples (10 μg) were separated on 4-20% SurePAGE™ precast gels (GenScript) and transferred to PVDF membranes (Millipore).

[0065] Membranes were blocked with QuickBlock™ Western Blocking Buffer (Beyotime) for 15 min at room temperature, and then incubated with specific primary antibodies as described in Table 1 overnight at 4°C.

[0066] Table 1

[0067]

[0068] Subsequently, membranes were incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (goat anti-rabbit IgG, HA1001, 1:50,000, HUABIO; goat anti-mouse IgG, HA1006, 1:20,000, HUABIO) for 1 h, and developed using an ECL chemiluminescence kit (Beyotime).

[0069] As shown in Fig. I, protein immunoblotting results showed that dimethyl succinate can significantly promote the expression of tooth differentiation-related proteins DSPP (as shown in Fig. II) and DMP1 (as shown in Fig. III) in the concentration range of 250 nM-1 μM, suggesting that this concentration can effectively induce tooth differentiation of dental pulp stem cells. Figure 3 Therefore, according to the experimental results, the addition of dimethyl succinate can increase the level of histone succinylation, and the use of pan-succinylation antibodies shows that dimethyl succinate can significantly enhance the level of histone succinylation and promote the transcriptional activation of tooth differentiation-related genes.

[0070] At the same time, as shown in Fig. I, the use of succinylation site antibodies shows that dimethyl succinate can promote the enhancement of H3K14 site succinylation, while the succinylation of H3K122, H3K79, and H2BK120 sites has no obvious change, indicating that dimethyl succinate, like Wnt3a, induces tooth differentiation of dental pulp stem cells by inducing enhancement of histone H3K14 succinylation. In addition, as can be seen from the figure, the direct addition of succinic acid does not have obvious enhancement effect, because succinic acid can start other signal pathways in the body and cannot directly enter the cell. Therefore, when using succinic acid as a dentin inducer, it can be prepared into a nanomaterial or directly introduced into the cell to play an inducing role.

[0071] Figure 4

[0072] ​​In the present application, since it is found that Wnt3a can improve the enhancement of H3K14 site succinylation, and the enhancement of H3K14 site succinylation can initiate the transcriptional activation of a large number of odontogenic genes, the inventors speculate that improving the enhancement of H3K14 site succinylation can improve the efficiency of odontogenic differentiation and induce odontogenic differentiation. In order to verify this guess, the present application uses dimethyl succinate to verify that dimethyl succinate can indeed induce stem cells to initiate odontogenic differentiation by improving the level of H3K14 site succinylation.

[0073] In addition, since succinic acid and its derivatives can induce protein succinylation enhancement in mechanism, and it has been verified by experiments that dimethyl succinate can improve the level of H3K14 site succinylation, therefore, small molecule substances with succinic acid group or capable of improving intracellular succinic acid level can all achieve the effect of inducing odontogenic differentiation.

[0074] In summary, the present application finds a new type of small molecule dentin inducer with high efficiency and can replace traditional protein preparations, which has important significance for promoting the clinical transformation of pulp dentin complex regeneration technology.

[0075] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A dentin-inducing agent that enhances histone succinylation, characterized in that, The dentin-inducing agent includes succinic acid or a succinic acid derivative; The succinic acid derivatives include succinates, succinamides, or pharmaceutically acceptable salts thereof.

2. The dentin-inducing agent according to claim 1, characterized in that, The dentin-inducing agent includes succinic acid and / or dimethyl succinate.

3. The use of the dentin inducer as described in claim 1 or 2 in the preparation of tooth differentiation-promoting products.

4. The application according to claim 3, characterized in that, The tooth differentiation-promoting products include any one or a combination of at least two of the following: pharmaceutical preparations, cell culture media, or implant materials.

5. The application according to claim 4, characterized in that, The concentration of dentin-inducing agent in the cell culture medium is 250 nM-1 μM.

6. The application according to claim 4, characterized in that, The implant material includes hydrogel materials and / or scaffold materials.

7. Application of succinic acid or succinic acid derivatives in the preparation of histone H3K14 succinylation inducers.

8. A dentin-inducing agent that inhibits desuccinylase function, characterized in that, The dentin inducer includes Sirt5 inhibitors and / or Sirt7 inhibitors.

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