Application of EHD3 and ATP6V1E1 as marker and therapeutic target for predicting progression of periodontal disease

Abstract

The invention is applicable to the technical field of biomedicine, and provides application of EHD3 and ATP6V1E1 as markers and therapeutic targets for predicting the progress of periodontal diseases. According to the method, non-invasive easily-obtained saliva samples of healthy people and patients with mild, moderate and severe periodontitis clinically diagnosed are collected, exosomes in the saliva samples are purified, and proteomics sequencing research is carried out. In the research process, the purified exosome is identified, and then proteomics detection is carried out. Through deep analysis of proteomics data, two key proteins, namely EHD3 and ATP6V1E1, are found, and the expression quantities of the two key proteins are continuously increased along with the progress of periodontitis from mild to moderate to severe. The two proteins can accurately reflect the progress of the periodontal disease, so that the two proteins can be used as biomarkers for predicting and diagnosing the progression of the periodontal disease at different stages, and meanwhile, potential targets are provided for effective treatment of the periodontal disease.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and focuses on the application of EHD3 and ATP6V1E1 as markers for predicting the progression of periodontal disease and as therapeutic targets. Background Art

[0002] Periodontal disease is one of the most common chronic diseases in the world. Currently, severe periodontitis has become the sixth most common disease, affecting 10.8% of the world's population. Untreated periodontal disease will cause progressive destruction of periodontal tissues and is the main factor leading to tooth loss. It may also cause various systemic diseases, seriously affecting the patient's quality of life. Therefore, there is an urgent need to develop sensitive and efficient new targets for early prediction of periodontal disease progression and targeted intervention treatment, which is of great significance for oral clinical applications.

[0003] At present, the diagnosis of periodontal disease mainly relies on clinical examination, imaging evaluation and the personal experience and skills of clinicians, but lacks quantitative indicators at the molecular level. In terms of treatment, a relatively unified "symptomatic treatment" strategy is adopted, lacking sensitive and effective treatment targets.

[0004] Salivary exosomes are extracellular vesicles with a diameter of 30-150 nm that exist in saliva. They contain a variety of bioactive substances and play an important role in pathological and physiological processes such as intercellular communication, immune response, and cell migration. Because they are easily available, non-invasive, and stable in nature, they can still maintain the integrity of the membrane structure and the stability of the contents after long-term storage, which shows broad application potential in non-invasive diagnosis and targeted therapy.

[0005] In view of this, the present invention separated and extracted exosomes from the saliva of healthy people and patients with different degrees of periodontal disease, performed protein sequencing analysis, and identified the potential application of EHD3 and ATP6V1E1 as markers for predicting the progression of periodontal disease and therapeutic targets. This discovery provides a new research direction and practical basis for the molecular prediction and targeted treatment of periodontal disease progression. Summary of the invention

[0006] The purpose of the present invention is to provide the use of EHD3 and ATP6V1E1 as markers and therapeutic targets for predicting the progression of periodontal disease, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: Application of substances for detecting EHD3 protein expression levels in the preparation of early periodontitis diagnosis products.

[0008] Use of substances that downregulate the expression level of EHD3 protein in the preparation of drugs for treating, alleviating or improving early periodontitis.

[0009] Application of substances for detecting ATP6V1E1 protein expression levels in the preparation of products for predicting the progression of advanced periodontitis.

[0010] Use of a substance that downregulates the expression level of ATP6V1E1 protein in the preparation of a drug for preventing, treating, alleviating or improving advanced periodontitis.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention identified two key proteins, EHD3 and ATP6V1E1, whose expression levels increase as periodontitis progresses from mild to moderate and then to severe. The expression levels of these two proteins can accurately reflect the course of periodontal disease. Therefore, under non-invasive conditions, they can be used as early predictive and diagnostic biomarkers that objectively reflect the progression of periodontal disease at the molecular level. At the same time, they also provide potential targets for the early and effective treatment of periodontal disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For exosome identification; A is the morphology of the extracted exosomes observed by transmission electron microscopy (JEM-2100, 120kV, magnification 30000X); B is the detection of exosome particle size by nanoparticle size analyzer; C is Western Blot identification of exosome markers CD9, CD81, and CD63.

[0013] Figure 2 The following are the results of proteomics quality control analysis of the samples; A is the number of matched peptide sequences and the number of identified proteins; B is the distribution of protein intensity values ​​of each sample; C is the principal component analysis of the relative quantitative values ​​of each sample, showing the similarities and differences between samples.

[0014] Figure 3 The following are the results of protein expression analysis of the samples; A is a Venn diagram showing the number of protein types detected in each group, as well as the specific expression and common expression of proteins in different groups; B is a heat map showing the expression patterns of 178 proteins detected in periodontitis patient samples as the periodontal disease progresses; C is a Venn diagram showing the differentially expressed proteins screened out between groups based on Log2FC>0.4; D is a violin plot showing the expression levels of the screened key proteins EHD3 and ATP6V1E1 in each group.

[0015] Figure 4 The bubble chart shows the enrichment analysis of the differentially expressed proteins between the mild periodontitis group (MiP) and the healthy group (HC) in the Reactome pathway.

[0016] Figure 5Functional enrichment analysis of differentially expressed proteins between moderate periodontitis group (MoP) and healthy group (HC) in GO-BiologicalProcess database.

[0017] Figure 6 Functional enrichment analysis of differentially expressed proteins between severe periodontitis group (SP) and healthy group (HC) in the GO-Cellular Component database.

[0018] Figure 7 Figure 3 shows the enrichment analysis of the differentially expressed proteins in the Reactome pathway between the severe periodontitis group (SP) and the healthy group (HC).

[0019] Figure 8 Figure 3 shows the enrichment analysis of the Reactome pathway for differentially expressed proteins between the severe periodontitis group (SP) and the mild periodontitis group (MiP).

[0020] Fig. 9 Figure 2 is the interaction network analysis diagram of differentially expressed proteins between the groups. The color depth represents the multiple of difference, and the circle size represents the number of interacting proteins. Figure 2 is the interaction network analysis of down-regulated proteins between the moderate periodontitis group (MoP) and the mild periodontitis group (MiP), and the circles in the figure represent down-regulated proteins. Figure 2 is the interaction network analysis of up-regulated proteins between the severe periodontitis group (SP) and the healthy group (HC), and the circles in the figure represent up-regulated proteins. DETAILED DESCRIPTION

[0021] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0022] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0023] Example 1: Purification and identification of salivary exosomes; 10 ml of saliva was collected from each volunteer in the healthy group (n=6), mild (n=4), moderate (n=3) and severe (n=4) periodontitis groups. After being diluted 1:1 with PBS solution, differential ultracentrifugation technology was used. Based on the difference in sedimentation coefficients between exosomes and other coexisting pollutants, cells were removed by centrifugation at 800g for 10 minutes, dead cells were removed by centrifugation at 2000g for 30 minutes, cell debris were removed by centrifugation at 12000g for 45 minutes, and exosome particles were sedimented at 100000g. Exosomes were successfully isolated from saliva.

[0024] The morphology of the extracted salivary exosomes was observed and identified using a transmission electron microscope. It was found that the extracted salivary exosomes were "tray" or "cup-shaped" with diameters ranging from 30 to 150 nm ( Figure 1 A in the middle). The particle size was detected by a nanoparticle size analyzer, and it was found that the particle size of the extracted salivary exosomes showed a clear single-peak distribution. The particle size of most exosomes was concentrated between 50nm and 100nm, which was a typical exosome nanoscale size feature ( Figure 1 At the same time, the extracted exosome proteins were separated by SDS-PAGE electrophoresis using Western Blot technology, and exosome markers (CD9, CD81, CD63) were identified using specific antibodies ( Figure 1 Middle C).

[0025] Example 2: Exosome proteomics sequencing and data quality control; 1% protease inhibitor was added to the extracted exosome samples, and ultrasonic lysis was performed. The protein concentration was determined using a BCA kit. An equal amount of protein from each sample was enzymatically hydrolyzed, and the peptides were desalted using a Strata X SPE column. The peptide solution was chromatographically separated and identified by mass spectrometry using an EASY-nLC1200 ultra-high performance liquid phase system and an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific). Data was collected using a data-independent scanning (DIA) program, and the obtained DIA data were processed using DIA-NN (v.1.8) and the Homo_sapiens_9606_SP_20231220.fasta database to obtain the identification results and abundance of peptides and proteins ( Figure 2 The principal component analysis (PCA) statistical method was used to evaluate the sample reproducibility ( Figure 2 (C) The results showed that the healthy group (HC) and mild periodontitis group (MiP) tended to be separated from the moderate periodontitis group (MoP) and severe periodontitis group (SP) as a whole, and the intra-group aggregation could reflect the repeatability of the samples.

[0026] Example 3: Screening of differentially expressed proteins; Proteins expressed in multiple samples in each group were selected for analysis, and the Venn diagram was used to screen for proteins detected in all periodontitis samples ( Figure 3 A) for subsequent analysis; a heat map was used to cluster the expression levels of the 178 proteins screened out. The results showed that the expression patterns of these proteins were divided into four different change trends with the progression of periodontal disease ( Figure 3In order to screen out the key proteins whose expression levels increased with the aggravation of periodontitis, the ratio of the mean values ​​of the proteins in the two groups of samples was used as the fold change (FC), and the relative quantitative values ​​of the proteins were compared between the groups using the T test, and the significance of the difference was determined by P value < 0.05. After logarithmic transformation, Log2FC>0.4 was used as the threshold, and the specific key proteins EHD3 and ATP6V1E1 that increased with the aggravation of periodontitis were obtained ( Figure 3 Middle C). The expression of EHD3 increased more significantly in mild to moderate periodontitis, and the expression of ATP6V1E1 increased more significantly in moderate to severe periodontitis, suggesting their main roles in different stages of periodontal disease progression ( Figure 3 Middle D).

[0027] EHD3 is a membrane-bound protein that is deeply involved in biological processes such as ciliary vesicle formation, endocytic transport, and cell-to-cell adhesion regulation, and plays an important role in immune disorders and intercellular communication. ATP6V1E1 can acidify and maintain the pH value in organelles. In some biological processes, it can target the cytoplasmic membrane, acidify the extracellular environment, and regulate the maturation of osteoclasts.

[0028] Example 4: Protein function and pathway enrichment cluster analysis; GO functional enrichment analysis and Reactome pathway enrichment analysis were performed on the differentially expressed proteins between different groups, and Fisher's exact test was used to calculate the significant P value to find the enrichment trend of the differentially expressed proteins between the groups in the functional type and pathway. In the comparison between the mild periodontitis group (MiP) and the healthy group (HC), the activation of the glucose metabolism pathway and the inflammatory response can be seen, and the activation of the non-classical β-catenin-independent Wnt signaling pathway is enriched, which suggests the activation of biological processes such as cell connection, cell migration, and cytoskeleton rearrangement ( Figure 4 In the comparison between the moderate periodontitis group (MoP) and the healthy group (HC), more active immune activation pathways and cell-cell junction regulation pathways were enriched ( Figure 5 ). The activation of these key functions and pathways indicates that in early mild and moderate periodontitis, high expression of salivary exosomal EHD3 protein regulates intercellular connections by activating the non-canonical Wnt pathway, promoting inflammatory infiltration of periodontal connective tissue and soft tissue destruction, leading to loss of periodontal tissue attachment.

[0029] In the comparison between the severe periodontitis group (SP) and the healthy group (HC), multiple functions related to the activation of V-type ATPase and the activation of the insulin signaling pathway were significantly enriched ( Figure 6 and Figure 7). The V-type ATPase family has a proton pump function, which can acidify and maintain the pH value in organelles. It can also target the plasma membrane, acidify the extracellular environment, and promote osteoclast maturation and differentiation. Insulin signals can promote the differentiation of osteoclast precursor cells and increase the number of osteoclasts. Existing literature has pointed out that the acidification effect of V-type ATPase can promote the maturation and release of insulin granules. These further indicate that in late-stage severe periodontitis, the high expression of salivary exosome ATP6V1E1 protein can target and activate osteoclast differentiation, while promoting the release of insulin signals, further increasing the number of osteoclasts, and playing a key role in the alveolar bone resorption process in the late stage of periodontitis. In addition, in the comparison between the severe periodontitis group (SP) and the mild periodontitis group (MiP), multiple apoptosis and cell death-related pathways were enriched, which is consistent with the aggravated tissue damage in severe periodontitis ( Figure 8 ).

[0030] Example 5: Protein interaction network analysis; By comparing with the STRING protein interaction network database, the differential protein interaction relationships were extracted according to the confidence score > 0.7 (high confidence). In the comparison between the moderate periodontitis group (MoP) and the mild periodontitis group (MiP), the interactions between the down-regulated differential proteins EPCAM, KRT18, and SPINT2 were obtained ( Fig. 9 In the comparison between the severe periodontitis group (SP) and the healthy group (HC), the upregulated differentially expressed proteins included the screened key protein ATP6V1E1 and the V-type ATPase family proteins ATP6V0D1, ATP6V0A4 and ATP6V1H ( Fig. 9 This further illustrates the major role of V-type ATPase, especially the key protein ATP6V1E1, in alveolar bone resorption and destruction in severe periodontitis.

[0031] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of substances for detecting EHD3 protein expression levels in the preparation of early periodontitis diagnosis products.

2. The use of substances that downregulate the expression level of EHD3 protein in the preparation of drugs for the treatment, relief or improvement of early periodontitis.

3. Application of substances for detecting ATP6V1E1 protein expression levels in the preparation of products for predicting the progression of advanced periodontitis.

4. Use of substances that downregulate the expression level of ATP6V1E1 protein in the preparation of drugs for preventing, treating, alleviating or improving advanced periodontitis.

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