Dentin inducer for enhancing histone succinylation and use thereof

Abstract

The present application provides a kind of dentin inducer for enhancing histone succinylation and its application, wherein the dentin inducer is dimethyl succinate.The present application finds that Wnt3a protein with clear odontogenic differentiation effect promotes odontogenic differentiation of dental pulp stem cells by increasing intracellular succinic acid content to induce histone succinic acid, and direct addition of succinic acid or succinic acid derivatives such as dimethyl succinate can play a Wnt3a replacement role, enhance stem cell histone succinylation and promote odontogenic differentiation of dental pulp stem cells.Furthermore, compared with Wnt3a protein class inducer, succinic acid or succinic acid derivative has the advantages of stable preparation and clear target, and is more suitable for clinical operation, which has important practical significance for promoting the clinical transformation of dental pulp dentin complex regeneration technology.

Description

Technical Field

[0001] This invention belongs to the field of dental pulp and dentin complex regeneration. Specifically, this invention relates to a dentin inducing agent that enhances histone succinylation and its application. Background Technology

[0002] Histone lysine succinylation (Ksuc), a newly discovered post-translational modification of histones, has attracted widespread attention from researchers in recent years. This modification alters the chemical environment of lysine residues by adding a succinyl group, thereby affecting the interaction between histones and DNA and profoundly impacting cell fate and function. Studies have shown that aberrant regulation of histone lysine succinylation plays a crucial role in the development and progression of cancer. Furthermore, research has indicated that histone lysine succinylation also plays an important role in hepatitis B virus infection.

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

[0004] In recent years, with the rapid development of stem cell biology and tissue engineering, stem cell-based regeneration strategies for the dental pulp-dentin complex have shown promising application prospects. Among them, dental pulp stem cells (DPSCs) have become a key focus of dental regenerative medicine research due to their wide availability, convenient acquisition, and multi-lineage differentiation potential.

[0005] Dentin-pulp regeneration is of significant clinical importance for maintaining tooth vitality, restoring tooth function, and extending tooth lifespan. Currently, it mainly relies on protein preparations such as Wnt3a and Wnt10a to induce the differentiation of dental pulp stem cells and promote dentin regeneration. These growth factors regulate stem cell proliferation and differentiation by activating specific signaling pathways, thereby promoting dentin formation.

[0006] However, these protein molecules face numerous technical obstacles in their application. Mechanistically, the molecular mechanisms by which these growth factors promote dentin regeneration are not fully understood, particularly their role in cellular metabolic regulation, which hinders the optimization and standardization of treatment efficacy. In terms of application, firstly, the inherent instability of protein molecules leads to rapid degradation in vivo, resulting in extremely low bioavailability and difficulty in maintaining sustained effective biological concentrations. Secondly, the delivery and localization of protein formulations are significantly limited, making it impossible to precisely control the target site and concentration gradient, thus affecting induction efficiency. Furthermore, the high cost of protein purification and preparation, along with complex production processes, severely restricts their widespread application.

[0007] Therefore, in order to overcome the limitations of existing protein preparations, improve treatment efficacy, and reduce treatment costs, this invention attempts to develop a novel dentin inducer with good stability, high bioavailability, a clear mechanism of action, and good cost-effectiveness by utilizing the succinylation mechanism. This has important practical significance for promoting the clinical translation of dental pulp-dentin complex regeneration technology. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a dentin inducer that enhances histone succinylation and its application.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a dentin inducer that enhances histone succinylation, said dentin inducer comprising succinic acid or a succinic acid derivative;

[0011] The succinic acid derivatives include succinates, succinic amides, or pharmaceutically acceptable salts thereof.

[0012] This invention discovers that the Wnt3a protein, which has a clear role in odontogenic differentiation, promotes odontogenic differentiation of dental pulp stem cells by inducing histone succinylation after increasing intracellular succinic acid content. Based on this, using Wnt3a as a positive control, this invention experimentally verified that succinic acid or succinic acid derivatives can exert the same effect as Wnt3a, significantly enhancing histone succinylation levels and thus promoting the transcriptional activation of odontogenic differentiation-related genes. In other words, succinic acid or succinic acid derivatives, by increasing succinic acid concentration to promote succinylation, can serve as a novel dentin inducer, replacing Wnt3a to enhance histone succinylation in stem cells while simultaneously promoting odontogenic differentiation of dental pulp stem cells. Succinylation refers to the process of covalently binding a succinyl group to an amino acid residue (mainly lysine K) through enzymatic or non-enzymatic methods.

[0013] In this invention, the succinic acid or succinic acid derivatives include small molecule substances such as succinic acid and dimethyl succinate that can provide succinyl groups and improve the succinylation level.

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

[0015] In a second aspect, the present invention provides the use of the dentin inducer as described in the first aspect in the preparation of tooth differentiation-promoting products.

[0016] As a preferred technical solution of the present invention, the tooth differentiation promoting product includes any one or a combination of at least two of the following: pharmaceutical preparations, cell culture media, or implant materials.

[0017] In this invention, the tooth differentiation-promoting products include various forms of biological products. Among them, succinic acid derivatives, such as dimethyl succinate, can be used directly, i.e., by placing dimethyl succinate directly into the root canal of a tooth, or by adding dimethyl succinate to a culture medium to treat stem cells in vitro, and then transplanting the stem cells into the root canal of a tooth. Furthermore, dimethyl succinate can also be combined with other drugs or excipients to form different types of implantable materials for placement in the root canal of a tooth. However, because succinic acid activates other signaling pathways in the body and cannot directly enter cells, it is necessary to use other auxiliary materials or combine it with other substances that can enter cells to successfully enter the cells and increase the concentration of succinic acid. For example, this invention can prepare succinic acid-containing nanoparticles or carry succinic acid through a scaffold and introduce it into cells.

[0018] As a preferred embodiment of the present invention, the concentration of the dentin inducer in the cell culture medium is 250 nM to 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 to be used as a dentin inducer, succinic acid needs to be prepared into nanoparticles or other forms that can enter the cells.

[0019] As a preferred embodiment of the present invention, the implant material comprises a hydrogel material and / or a scaffold material; the hydrogel material or scaffold material is mainly used to carry succinic acid and introduce it into cells.

[0020] Thirdly, the present invention also provides the use of succinic acid or succinic acid derivatives in the preparation of histone H3K14 succinylation inducers.

[0021] This invention uses dimethyl succinate as an example to experimentally verify the target of succinic acid or succinic acid derivatives. Dimethyl succinate induces odontogenic differentiation by enhancing histone succinylation, thereby inducing the transcriptional activation of numerous key odontogenic genes. More specifically, it induces odontogenic differentiation in stem cells by enhancing succinylation at the H3K14 site. Prior to this, no related research had proposed a theory based on enhanced histone succinylation to induce odontogenic differentiation; this invention clarifies that this theory is one of the key mechanisms and targets for inducing odontogenic differentiation in stem cells.

[0022] Fifthly, the present invention also provides a dentin inducer that inhibits the function of desuccinylase, said dentin inducer comprising Sirt5 inhibitors and / or Sirt7 inhibitors.

[0023] Since enhancing histone succinylation can induce odontogenic differentiation of stem cells, small molecule preparations that inhibit the function of desuccinylation enzymes in dental pulp stem cells can also enhance histone succinylation, including small molecule drugs such as Sirt5 inhibitors (NRD167, etc.) and Sirt7 inhibitors (SIRT7 inhibitor 97491, etc.).

[0024] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. This invention first clarifies that succinic acid or succinic acid derivatives can induce odontogenic differentiation of dental pulp stem cells, and the induction effect is similar to that of the positive control Wnt3a, which is highly efficient and stable; moreover, inorganic salt components have the advantages of formulation stability and clear target compared with protein preparations, making them more suitable for clinical operation.

[0027] 2. This invention confirms that succinic acid or succinic acid derivatives induce the transcriptional activation of a large number of key odontogenic genes by enhancing histone succinylation, thereby inducing odontogenic differentiation; specifically, it provides enhanced succinylation at the H3K14 site, thereby inducing stem cells to initiate odontogenic differentiation. Attached Figure Description

[0028] Figure 1 The image shows the experimental results of H3K14su CUT&Tag after Wnt3a induction in Example 1.

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

[0030] Figure 3 The figures show a comparison of the expression levels of odontogenic differentiation-related proteins in dental pulp stem cells of each experimental group and the control group in Example 2. Figure I is a gel plot of protein expression levels; Figure II is a bar chart comparing the relative expression levels of DSPP protein; and Figure III is a bar chart comparing the relative expression levels of DMP1 protein.

[0031] Figure 4 This is a comparison of the succinylation levels of histones H3 and H2B after induction in each experimental group and control group in Example 2. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0033] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0034] Example 1: Cell grouping culture

[0035] Example 1: Investigating transcriptional activation sites of genes related to odontogenesis differentiation

[0036] It is known that Wnt3a can induce odontogenic differentiation. In order to further explore its mechanism of action, this embodiment studies its related activation sites through CUT&Tag experiments.

[0037] (1) Cell culture

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

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

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

[0041] (2) CUT & Tag Experiment

[0042] The Procell anti-mycoplasma reagent was used to completely eliminate mycoplasma contamination, and the decontamination effect was verified by the Yeasen MycAway™ Plus-Color one-step detection kit.

[0043] In the experimental procedure, cells were coupled to magnetic beads coated with capreomycin A, and cell fixation was achieved through surface glycoprotein-mediated fixation. Digital mycin treatment permeated the cell membrane, followed by the introduction of specific antibodies to achieve precise binding to the target protein.

[0044] The highly active pG-Tn5 / pA-Tn5 transposase was used to precisely locate the Tn5 enzyme through the interaction of the G / A domains of the Fc region protein. In an optimized buffer system, the transposase simultaneously performed chromatin fragmentation and Illumina sequencing adapter ligation. DNA fragments were purified and extracted using phenol-chloroform, and then amplified and concentrated by PCR using adapter-specific primers. Library quality was assessed using an Agilent 2100 bioanalyzer and Qubit quantitative analysis, followed by high-throughput sequencing on the Illumina NovaSeq platform.

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

[0046] like Figure 1 As shown, using CUT&Tag experiments, enhanced H3K14 succinylation levels were found to be accompanied by significant enrichment of H3K14su signal in the gene promoter region, suggesting that it induces transcriptional activation of differentiation-related genes in dental pulp stem cells, accelerating their odontogenic differentiation process. In other words, the odontogenic differentiation induced by Wnt3a is related to its ability to enhance H3K14 succinylation levels.

[0047] Example 2: Verification of the induction effect of succinylated derivatives

[0048] As demonstrated in Example 1, Wnt3a can enhance the succinylation level of H3K14. Due to the limitations of protein preparations and the fact that succinic acid and its derivatives often induce enhanced protein succinylation, this example attempts to verify whether succinylation derivatives can achieve the effect of enhancing the succinylation level of H3K14, 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), dimethyl succinate group (E-Succ).

[0052] The control group was induced by odontogenic differentiation medium, the succinic acid group was induced by odontogenic differentiation medium supplemented with 1 μM succinic acid (Sigma), and the dimethyl succinate groups (a total of five groups) were induced by odontogenic differentiation medium supplemented with gradient concentrations of dimethyl succinate (Sigma), as follows:

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

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

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

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

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

[0058] (3) Verification Experiment

[0059] 1. Alizarin Red S staining: To assess odontogenic differentiation, calcium deposition was analyzed at predetermined time points using Alizarin Red S solution (Cyagen).

[0060] like Figure 2 As shown, the results of alizarin red staining indicate that the addition of 250 nM-1 μM dimethyl succinate can enhance the formation of calcium nodules, but there is no significant enhancing effect above this concentration.

[0061] 2. Determination of expression levels of odontogenesis-related proteins and histone succinylation sites.

[0062] Total cellular proteins were extracted using RIPA lysis buffer (Beyotime) containing a mixture of protease inhibitors (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 gel (GenScript) and transferred to a PVDF membrane (Millipore).

[0065] The membrane was blocked at room temperature for 15 minutes using QuickBlock™ Western blocking buffer (Beyotime), and then incubated overnight at 4°C with the specific primary antibody as described in Table 1.

[0066] Table 1

[0067]

[0068] Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG, HA1001, 1:50,000, HUABIO; goat anti-mouse IgG, HA1006, 1:20,000, HUABIO) for 1 hour and then developed using an ECL chemiluminescence kit (Beyotime).

[0069] like Figure 3 As shown in Figure I, the protein immunoblotting results show that dimethyl succinate can significantly promote the expression of odontogenic differentiation-related proteins DSPP (as shown in Figure II) and DMP1 (as shown in Figure III) within the concentration range of 250 nM to 1 μM, suggesting that this concentration can effectively induce odontogenic differentiation of dental pulp stem cells.

[0070] Therefore, the experimental results show that the addition of dimethyl succinate increases the level of histone succinylation. Using pansuccinylation antibody, it was found that dimethyl succinate can significantly enhance the level of histone succinylation and promote the transcriptional activation of odontogenic differentiation-related genes.

[0071] At the same time, such as Figure 4 As shown, using antibodies against succinylation sites, dimethyl succinate was found to promote enhanced succinylation at the H3K14 site, while succinylation at H3K122, H3K79, and H2BK120 sites showed no significant change. This confirms that dimethyl succinate, like Wnt3a, induces odontogenic differentiation of dental pulp stem cells by enhancing histone H3K14 succinylation. Furthermore, the figure shows that direct addition of succinic acid did not significantly enhance the effect. This is because succinic acid activates other signaling pathways in vivo and cannot directly enter cells. Therefore, when using succinic acid as a dentin inducer, it is advisable to prepare it as a nanomaterial or directly introduce it into cells to exert its inducing effect.

[0072] In this invention, since Wnt3a was found to enhance succinylation at the H3K14 site, and enhanced succinylation at the H3K14 site can initiate transcriptional activation of a large number of odontogenic genes, the inventors hypothesized that enhancing succinylation at the H3K14 site could improve odontogenic differentiation efficiency and induce odontogenic differentiation. To verify this hypothesis, this invention used dimethyl succinate for verification, and confirmed that dimethyl succinate can indeed induce stem cells to initiate odontogenic differentiation by increasing the level of succinylation at the H3K14 site.

[0073] Furthermore, since succinic acid and its derivatives can induce enhanced protein succinylation, and it has been experimentally verified that dimethyl succinate can increase the succinylation level at the H3K14 site, small molecules with succinic acid groups or those that can increase intracellular succinic acid levels can all achieve the effect of inducing odontogenic differentiation.

[0074] In summary, this invention has discovered a novel, highly efficient small-molecule dentin inducer that can replace traditional protein preparations, which is of great significance for promoting the clinical translation of dental 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 invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of dimethyl succinate in the preparation of a tooth differentiation promoting preparation.

2. Use according to claim 1, characterized in that, The tooth differentiation promoting preparation comprises any one or a combination of at least two of a pharmaceutical preparation, a cell culture medium or an implant material.

3. Use according to claim 2, characterized in that, The concentration of dimethyl succinate in the cell culture medium is from 250 nM to 1 μM.

4. Use according to claim 2, characterized in that, The implant material comprises a hydrogel material and / or a scaffold material.

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