Application of ENTR1 gene in preparation of medicine for treating periodontal tissue inflammation

Abstract

The invention relates to application of an ENTR1 gene in preparation of a medicine for treating periodontal tissue inflammation. The nucleotide sequence of the ENTR1 gene is as shown in SEQ ID No. 1. The application of the ENTR1 gene in preparation of the medicine for treating the periodontal tissue inflammation comprises the following two aspects: (1) applying the ENTR1 gene as an action target to preparation of the medicine for treating the periodontal tissue inflammation; and (2) applying the ENTR1 gene as an action target to screening of drugs for treating periodontal tissue inflammation. Through construction of a periodontitis mouse model, it is found that the expression level of the ENTR1 gene in a periodontitis mouse is remarkably reduced, the expression levels of inflammatory factors TNF-alpha, IL-6 and iNOS mRNA are remarkably increased, meanwhile, infiltration of M1 type macrophages in periodontal tissue can be effectively reduced through overexpression of the ENTR1 gene, and the expression level of the M1 type macrophages in the periodontal tissue can be effectively reduced. And by injecting an ENTR1 overexpression adenovirus vector and a control adenovirus empty vector, the occurrence and development of mouse periodontitis can be obviously inhibited by the specific overexpression ENTR1 gene.

Description

Technical Field

[0001] The invention relates to application of ENTR1 gene in preparing medicine for treating periodontal tissue inflammation, and belongs to the field of molecular biology and biotechnology. Background Art

[0002] Periodontitis is an inflammatory disease with periodontal pocket formation, attachment loss, and alveolar bone resorption as its main clinical manifestations. It is the main cause of tooth loss in adults. The pathogenesis of periodontitis is closely related to the host's autoimmune inflammatory response. Macrophages, as an important component of the host's innate immune system, play an important role in the occurrence and development of periodontitis. After being stimulated by different types of stimulation, primitive macrophages (M0) can polarize into two different phenotypes: classical pro-inflammatory (M1) or anti-inflammatory (M2) in response to local physiological or pathological conditions. M1 macrophages have pro-inflammatory effects and can be activated by lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), etc., to secrete high levels of pro-inflammatory chemokines and cytokines, including interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), etc. M2 macrophages have anti-inflammatory effects and are mostly induced by interleukin-4 (IL-4) or interleukin-13 (IL-13). They release anti-inflammatory factors such as interleukin-10 (IL-10) and promote the repair of damaged tissues. During periodontitis, macrophages undergo corresponding phenotypic transformations that affect disease progression. Activated M1 macrophages can cause periodontal tissue destruction. In contrast, M2 macrophages promote tissue repair and enhance periodontal tissue regeneration.

[0003] ENTR1 (Endosome-associated Trafficking Regulator 1), also known as Sdccag3 (Serologically Defined Colon Cancer Antigen-3), is a widely expressed endosomal protein that plays an important role in biological behaviors such as protein transport, ciliogenesis, apoptosis, and mitosis. Existing studies have found that ENTR1 has the ability to regulate the osteogenic / adipogenic bidirectional differentiation of bone marrow mesenchymal stem cells (BMSCs) and promote new bone formation around implants in experimental hyperlipidemia rats, but its effect on bone loss caused by periodontal inflammation needs further study. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of ENTR1 gene in the preparation of a drug for treating periodontal tissue inflammation.

[0005] The technical solution of the present invention is as follows:

[0006] The use of ENTR1 gene in preparing a drug for treating periodontal tissue inflammation, wherein the nucleotide sequence of the ENTR1 gene is shown in SEQ ID No.1.

[0007] Preferably, according to the present invention, the use of the ENTR1 gene in the preparation of a drug for treating periodontal tissue inflammation includes the following two aspects:

[0008] (1) Use the ENTR1 gene as a target for the preparation of drugs for the treatment of periodontal inflammation;

[0009] (2) Use the ENTR1 gene as a target for screening drugs for the treatment of periodontal tissue inflammation.

[0010] Preferably according to the present invention, the ENTR1 gene is used as a target for the preparation of a drug for treating periodontal tissue inflammation, which means: using the ENTR1 gene as a target for drugs or preparations, increasing the expression level of the ENTR1 gene in periodontal tissues, and developing drugs or preparations for periodontal tissue inflammation.

[0011] Preferably according to the present invention, the use of the ENTR1 gene as a target for screening drugs for treating periodontal tissue inflammation means: using the ENTR1 gene as a target for drugs or preparations, screening the drugs or preparations to find drugs or preparations that can promote the expression of the ENTR1 gene in periodontal tissues as alternative drugs or preparations for treating periodontal tissue inflammation.

[0012] Preferably according to the present invention, the drugs for treating periodontal tissue inflammation include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or viruses.

[0013] Further preferably, the virus is an ENTR1 overexpression adenovirus vector, the active ingredient of which contains the nucleotide sequence shown in SEQ ID No. 1, and is specifically prepared by co-transfecting the host cells with the recombinant expression plasmid and the packaging system, and the infiltration of M1 macrophages in the periodontal tissue is reduced by overexpressing the ENTR1 gene to treat periodontal tissue inflammation.

[0014] Preferably according to the present invention, the drug for treating periodontal tissue inflammation further contains a pharmaceutically acceptable excipient.

[0015] More preferably, the excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose and water.

[0016] Preferably according to the present invention, the drug for treating periodontal tissue inflammation is in the form of tablets, pills, powder or injection.

[0017] Technical features and beneficial effects of the present invention:

[0018] 1. The present invention constructs a periodontitis mouse model and finds that the expression level of ENTR1 gene in periodontitis mice is significantly downregulated, while the expression levels of inflammatory factors TNF-α, IL-6 and iNOS mRNA are significantly upregulated. At the same time, overexpression of ENTR1 gene can effectively reduce the infiltration of M1 macrophages in periodontal tissues, thereby providing a theoretical basis for preparing or screening drugs or preparations that promote ENTR1 gene expression as alternative drugs or preparations for the treatment of periodontitis.

[0019] 2. The present invention prepared an ENTR1 overexpression adenovirus vector. By injecting the ENTR1 overexpression adenovirus vector and a control adenovirus empty vector, it was found that specific overexpression of the ENTR1 gene in the periodontal tissue of mice can significantly inhibit the occurrence and development of periodontitis in mice.

[0020] 3. The present invention uses the ENTR1 gene as a target to prepare or screen drugs for treating periodontitis. Drug screening is mainly aimed at unknown drugs, and the drugs are acted on the target gene. The drugs for treating periodontitis are screened according to whether the drugs can promote the expression of the target gene; drug preparation is mainly based on the target gene, and drugs for treating periodontitis are prepared or constructed in a targeted manner to promote the expression of the target gene; the screened or prepared drugs are of great significance in the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of qRT-PCR results of relevant molecular expression levels in the experimental periodontitis group and the healthy control group.

[0022] Figure 2 Schematic diagram of the hematoxylin-eosin staining results of periodontal tissues in the experimental periodontitis group and the healthy control group.

[0023] Figure 3 Schematic diagram of the immunohistochemical staining results of periodontal tissues in the experimental periodontitis group and the healthy control group.

[0024] Figure 4 Schematic diagram of immunofluorescence staining results of periodontal tissues in the experimental periodontitis group and the healthy control group.

[0025] Figure 5 Schematic diagram of the recombinant expression plasmid structure used to construct an ENTR1 overexpression adenovirus vector.

[0026] Figure 6 Schematic diagram of qRT-PCR detection results after local injection of ENTR1 overexpressing adenovirus vector into periodontal tissue.

[0027] Figure 7Schematic diagram of the hematoxylin-eosin staining results of periodontal tissue after ENTR1 overexpression.

[0028] Figure 8 Schematic diagram of the immunohistochemical staining results of periodontal tissue after ENTR1 overexpression.

[0029] Fig. 9 Schematic diagram of the immunofluorescence staining results of periodontal tissue after ENTR1 overexpression. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with examples, which are only used to illustrate the present invention but not to limit the scope of protection of the present invention.

[0031] Unless otherwise specified, the drugs and reagents involved in the examples are all common commercially available products; the experimental operations involved in the examples are all carried out according to routine operations in the art unless otherwise specified.

[0032] The pAdM-FH-GFP plasmid vector was purchased from Shandong Weizhen Biotechnology Co., Ltd.

[0033] Example 1: Establishment of a ligature-induced periodontitis model in mice

[0034] Ten male, 8-week-old c57BL / 6J mice were randomly divided into two groups, the first group was a healthy control group, and the second group was a ligature-induced experimental periodontitis group. Under inhalation anesthesia, a 5-0 ligature was inserted between the first and second molars of the mice in the experimental periodontitis group, and the mice were housed under specific pathogen-free conditions with free access to food and water for 7 days to induce experimental periodontitis and bone resorption, thus obtaining a periodontitis mouse model.

[0035] Seven days after the periodontitis mouse model was successfully established, the two groups of mice were euthanized by overdose injection of sodium pentobarbital. The periodontal tissues around the maxillary bones of the two groups of mice were taken and stored in liquid nitrogen and 4% paraformaldehyde, respectively, for later detection.

[0036] Example 2: Effect of periodontitis on the expression of inflammatory factors in periodontal tissues of mice

[0037] The qRT-PCR method was used to detect the expression levels of ENTR1 gene, inflammatory factors TNF-α and IL-6 mRNA in the periodontal tissues of mice in the healthy control group (control) and experimental periodontitis group (periodontitis) in Example 1. The test results are as follows: Figure 1 shown.

[0038] Depend on Figure 1It can be seen that compared with the healthy control group, the expression level of ENTR1 gene in the periodontal tissue of mice in the experimental periodontitis group was significantly downregulated, while the expression levels of inflammatory factors TNF-α and IL-6 mRNA were significantly upregulated, which indicates that periodontitis promotes the expression of proinflammatory cytokines in the periodontal tissue of mice, and the significantly increased expression level of proinflammatory cytokines in the periodontal tissue of mice is a significant feature of the occurrence and development of periodontitis.

[0039] Example 3: Tissue embedding and sectioning and hematoxylin-eosin staining (H&E staining)

[0040] The periodontal tissues of the healthy control group (control) and the experimental periodontitis group (periodontitis) mice obtained in Example 1 were fixed with 4% paraformaldehyde, decalcified in EDTA (pH=7.5) for 6 weeks, dehydrated in a gradient of 50% to 100% ethanol and embedded in paraffin, cut into 4 μm thick slices with a paraffin slicer, flattened and dried to obtain paraffin sections.

[0041] Paraffin sections were dewaxed and rehydrated with graded ethanol, and treated with high-definition constant stain pretreatment solution for 1 minute. The sections were placed in hematoxylin stain solution for 4 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue reversion solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1 minute, stained in eosin stain solution for 15 seconds, dehydrated and sealed, and examined under a microscope. Image acquisition and analysis were performed. The results were as follows: Figure 2 shown.

[0042] Depend on Figure 2 It can be seen that compared with the healthy control group (control), the alveolar bone height of the periodontal tissue of mice in the experimental periodontitis group was significantly reduced, indicating that periodontal inflammation will increase alveolar bone loss.

[0043] Example 4, Immunohistochemical staining (IHC staining)

[0044] The paraffin sections in Example 3 were dewaxed and rehydrated with graded ethanol, and incubated with antigen extraction solution. The sections were incubated in 3% hydrogen peroxide solution at room temperature for 25 minutes, and then incubated with IL-1β antibody in a humidified box at 4°C overnight. The next day, the tissue was covered with a secondary antibody (HRP labeled) of the same species as the primary antibody and incubated at room temperature for 50 minutes. After color development with DAB color developing solution and a brown precipitate, the cell nuclei were counterstained with hematoxylin for 3 minutes. After dehydration and sealing, images were collected under a microscope. The results are as follows: Figure 3 shown.

[0045] Depend on Figure 3It can be seen that compared with the healthy control group (control), the expression level of the inflammatory factor IL-1β in the periodontal tissue of mice in the experimental periodontitis group (periodontitis) was significantly increased, indicating that periodontal inflammation will increase the expression of IL-1β in periodontal tissue.

[0046] Example 5, Immunofluorescence staining (IF staining)

[0047] The paraffin sections in Example 3 were dewaxed and rehydrated with graded ethanol for antigen retrieval. The sections were then placed flat in a light-proof humidified box, 3% hydrogen peroxide solution was added, and the sections were incubated at room temperature in the dark for about 25 minutes to block endogenous peroxidase. After washing with PBS buffer, serum was added and blocked at room temperature for 30 minutes. Gently shake off the blocking solution, add CD68 primary antibody to the sections, and the sections were placed in a light-proof humidified box and incubated at 4°C overnight. Then add the corresponding species HRP-labeled secondary antibody and incubate at room temperature for 50 minutes. The sections were placed in a light-proof humidified box, the corresponding TSA was added, and incubated at room temperature in the dark for 10 minutes. Serum was added and blocked at room temperature for 30 minutes. Gently shake off the blocking solution, add CD86 primary antibody to the sections, and the sections were placed in a light-proof humidified box and incubated at 4°C overnight. Add the corresponding species HRP-labeled secondary antibody and incubate at room temperature for 50 minutes. DAPI counterstaining of cell nuclei: After the slices are slightly dried, place them flat in a light-proof humidified box, add DAPI staining solution, and incubate at room temperature in a dark place for 10 minutes. After the slices are slightly dried, add autofluorescence quencher for 5 minutes and rinse with running water for 10 minutes. Seal the slices with anti-fluorescence quenching sealing agent and collect images with a fluorescence microscope. The results are as follows: Figure 4 shown.

[0048] Depend on Figure 4 It can be seen that compared with the healthy control group, the periodontal tissues of mice in the experimental periodontitis group (periodontitis) showed a significant increase in CD86 + / CD68 + The proportion of cells increased significantly, indicating that periodontal inflammation increases the number of CD86 + / CD68 + proliferation.

[0049] Example 6. Preparation of ENTR1 overexpression adenoviral vector

[0050] Target gene: ENTR1 gene, as shown in SEQ ID No. 1;

[0051] Restriction site: AsisI / MluI;

[0052] Vector tag: C-terminal fusion HA protein tag;

[0053] Vector resistance: Amp, resistance concentration is 100 μg / ml;

[0054] Carrier map: Figure 5 shown.

[0055] The ENTR1 gene nucleic acid sequence was subcloned into the pAdM-FH-GFP plasmid by restriction digestion-ligation-transformation to obtain a positive clone, and then sequencing was performed to confirm the correctness of the inserted fragment to obtain the recombinant expression plasmid of the ENTR1 gene. The sequencing primer sequence is as follows:

[0056] 5' end sequencing primer: 5'-CGCAAATGGGCGGTAGGCGTG-3',

[0057] 3' end sequencing primer: 5'-GGAAAGGACAGTGGGAGTG-3'.

[0058] Then, the recombinant expression plasmid and the packaging system are co-transfected into the host cell to obtain the ENTR1 overexpression adenovirus vector. The reagents used in the enzyme cleavage-ligation-transformation, packaging system and transfection process are all conventional reagents, and the enzyme cleavage-ligation-transformation, packaging system and transfection method are all publicly available conventional methods and can be performed according to the prior art.

[0059] Example 7: Therapeutic effect of ENTR1 overexpressing adenovirus vector on periodontal tissue inflammation

[0060] 1. Twenty male, 8-week-old c57BL / 6J mice were randomly divided into 4 groups, wherein the mice in groups 1 and 2 were injected with a control adenovirus empty vector (purchased from Shandong Weizhen Biotechnology Co., Ltd.) in the periodontal tissue, and the mice in groups 3 and 4 were injected with an ENTR1 overexpression adenovirus vector in the periodontal tissue. Then, the mice in groups 1 and 3 after injection were used to construct a ligation-induced periodontitis mouse model according to the method described in Example 1, and the mice in groups 2 and 4 were not treated. The 4 groups were respectively recorded as the overexpression negative control group (Adv-Con), the ENTR1 overexpression group (Adv-ENTR1), the ENTR1 overexpression negative control combined with ligation-induced periodontitis group (Adv-Con+lig), and the ENTR1 overexpression combined with ligation-induced periodontitis group (Adv-ENTR1+lig). Seven days after the successful modeling, the periodontal tissues of the 4 groups of mice were obtained by the method described in Example 1.

[0061] 2. Periodontal tissue samples of the above four groups of mice were taken and the expression of ENTR1 gene was detected by qRT-PCR. The results are as follows: Figure 6 shown.

[0062] Depend on Figure 6 It can be seen that compared with the Adv-Con group and Adv-Con+lig group, the expression level of ENTR1 gene in periodontal tissue of the Adv-ENTR1 group and Adv-ENTR1+lig group was significantly upregulated.

[0063] 3. The periodontal tissue samples of the above four groups of mice were subjected to H&E staining according to the method described in Example 3. The results are as follows: Figure 7 shown.

[0064] Depend on Figure 7 It can be seen that compared with the Adv-Con+lig group, the degree of alveolar bone loss in the Adv-ENTR1+lig group was significantly reduced.

[0065] 4. The periodontal tissues of the above four groups of mice were subjected to immunohistochemical staining according to the method described in Example 4. The results are as follows: Figure 8 shown.

[0066] Depend on Figure 8 It can be seen that after injection of ENTR1 overexpression adenovirus vector, the expression of IL-1β in the periodontal tissue of periodontitis mice was reduced, indicating that ENTR1 overexpression adenovirus vector can reduce the expression of proinflammatory cytokines.

[0067] 5. The periodontal tissues of the above four groups of mice were subjected to immunofluorescence staining according to Example 5. The results are as follows: Fig. 9 shown.

[0068] Depend on Fig. 9 It can be seen that compared with the Adv-Con+lig group, the CD86 + / CD68 + The number of cells increased significantly, indicating that the injection of ENTR1 overexpression adenovirus vector can reduce the infiltration of M1 macrophages in periodontal tissues. It was further confirmed that ENTR1 overexpression adenovirus vector can inhibit the polarization of M1 macrophages in periodontal tissues and inflammation of periodontal tissues in mice with periodontitis.

[0069] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. The use of ENTR1 gene in the preparation of a drug for treating periodontal tissue inflammation, characterized in that: The nucleotide sequence of the ENTR1 gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The use of the ENTR1 gene in preparing a drug for treating periodontal tissue inflammation includes the following two aspects: (1) Use the ENTR1 gene as a target for the preparation of drugs for the treatment of periodontal inflammation; (2) Use the ENTR1 gene as a target for screening drugs for the treatment of periodontal tissue inflammation.

3. The use according to claim 2, characterized in that The use of the ENTR1 gene as a target for the preparation of a drug for treating periodontal tissue inflammation means: using the ENTR1 gene as a target for a drug or preparation, increasing the expression level of the ENTR1 gene in periodontal tissue, and developing a drug or preparation for periodontal tissue inflammation.

4. The use according to claim 2, characterized in that The use of the ENTR1 gene as a target for screening drugs for treating periodontal tissue inflammation means: using the ENTR1 gene as a target for drugs or preparations, screening the drugs or preparations to find drugs or preparations that can promote the expression of the ENTR1 gene in periodontal tissues as alternative drugs or preparations for treating periodontal tissue inflammation.

5. The use according to claim 1, characterized in that The drugs for treating periodontal tissue inflammation include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or viruses.

6. The use according to claim 4, characterized in that The virus is an ENTR1 overexpression adenovirus vector, the active ingredient of which contains the nucleotide sequence shown in SEQ ID No. 1, and is specifically prepared by co-transfecting a host cell with a recombinant expression plasmid and a packaging system. The virus overexpresses the ENTR1 gene to reduce the infiltration of M1 macrophages in periodontal tissues, thereby treating periodontal tissue inflammation.

7. The use according to claim 1, characterized in that The drug for treating periodontal tissue inflammation also contains a pharmaceutically acceptable excipient.

8. The use according to claim 7, characterized in that The excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose and water.

9. The use according to claim 5, characterized in that The medicine for treating periodontal tissue inflammation is in the form of tablets, pills, powders or injections.

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