Use of EHD3 and ATP6V1E1 as markers for predicting the progression of periodontal disease and therapeutic targets

Abstract

The present invention is applicable to the field of biomedical technologies and provides the use of EHD3 and ATP6V1E1 as markers for predicting the progression of periodontal disease and therapeutic targets. The present invention collects non-invasive and easily obtainable saliva samples from healthy individuals and patients clinically diagnosed with mild, moderate, and severe periodontitis, purifies the exosomes therein, and conducts proteomic sequencing studies. During the research process, the purified exosomes are identified, and then proteomic detection is carried out. By deeply analyzing the proteomic data, the present invention discovers two key proteins, namely EHD3 and ATP6V1E1, whose expression levels continuously increase as periodontitis progresses from mild to moderate to severe. These two proteins can accurately reflect the process of periodontal disease, and thus can be used as predictive and diagnostic biomarkers for the progression of different stages of periodontal disease, and also provide potential targets for the effective treatment of periodontal disease.

Description

Technical Field

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

[0002] Periodontal disease is one of the most common chronic diseases globally. Currently, severe periodontitis has become the sixth most common disease, affecting 10.8% of the global population. Untreated periodontal disease can cause progressive damage to periodontal tissues, being the main factor leading to tooth loss. At the same time, it may trigger various systemic diseases, seriously affecting the quality of life of patients. Therefore, there is an urgent need to develop sensitive and efficient new targets for early prediction of the progression of periodontal disease and targeted intervention treatment, which is of great significance for oral clinical applications.

[0003] Currently, the diagnosis of periodontal disease mainly relies on clinical examinations, imaging evaluations, as well as the personal experience and techniques 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 diameters ranging from 30 to 150 nm present in saliva, containing various bioactive substances, and playing important roles in pathophysiological processes such as intercellular communication, immune response, and cell migration. Due to their easy accessibility, non-invasiveness, and stable nature, they can still maintain the integrity of the membrane structure and the stability of the inclusions after long-term storage, showing broad application potential in non-invasive diagnosis and targeted therapy.

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

[0006] The purpose of the present invention is to provide the application 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 art.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] Application of a substance for detecting the expression level of EHD3 protein in the preparation of a diagnostic product for early periodontitis.

[0009] Application of a substance for down-regulating the expression level of EHD3 protein in the preparation of a drug for treating, alleviating or improving early periodontitis.

[0010] Use of a substance for detecting the expression level of ATP6V1E1 protein in the preparation of a product for predicting the progression of advanced periodontitis.

[0011] Use of a substance for down-regulating the expression level of ATP6V1E1 protein in the preparation of a drug for preventing, treating, alleviating or improving advanced periodontitis.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The present invention has identified two key proteins, EHD3 and ATP6V1E1, whose expression levels continuously increase as periodontitis progresses from mild to moderate to severe. The expression levels of these two proteins can accurately reflect the progression of periodontal disease. Therefore, under non-invasive conditions, they can be used as early prediction 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

[0014] Figure 1 For exosome identification; wherein A shows the morphology of the extracted exosomes observed by transmission electron microscopy (JEM-2100, 120 kV, magnification 30000X); B shows the particle size of exosomes detected by a nanoparticle sizer; C shows the identification of exosome markers CD9, CD81, and CD63 by Western Blot.

[0015] Figure 2 For the results of proteomics quality control analysis of samples; wherein A shows the number of matched peptide sequences and the number of identified proteins; B shows the distribution of protein intensity values in each sample; C shows the principal component analysis of relative quantification values of each sample, demonstrating the similarities and differences between samples.

[0016] Figure 3 For the results of protein expression analysis of samples; wherein A shows a Venn diagram showing the number of protein species detected in each group, and the specific expression and co-expression of proteins in different groups; B shows a heat map showing the expression patterns of 178 proteins screened and detected in periodontitis patient samples during the progression of periodontal disease; C shows a Venn diagram showing the differentially expressed proteins screened based on Log2FC>0.4 between groups; D shows a violin plot showing the expression levels of the key proteins EHD3 and ATP6V1E1 screened in each group.

[0017] Figure 4 For a bubble plot showing the enrichment analysis of differentially expressed proteins in the Reactome pathway by comparing the mild periodontitis group (MiP) with the healthy control group (HC).

[0018] Figure 5The functional enrichment analysis of differentially expressed proteins in the comparison between the moderate periodontitis group (MoP) and the healthy group (HC) in the GO-Biological Process database.

[0019] Figure 6 The functional enrichment analysis of differentially expressed proteins in the comparison between the severe periodontitis group (SP) and the healthy group (HC) in the GO-Cellular Component database.

[0020] Figure 7 The Reactome pathway enrichment analysis of differentially expressed proteins in the comparison between the severe periodontitis group (SP) and the healthy group (HC).

[0021] Figure 8 The Reactome pathway enrichment analysis of differentially expressed proteins in the comparison between the severe periodontitis group (SP) and the mild periodontitis group (MiP).

[0022] Figure 9 The interaction network analysis diagram of differential proteins among groups. The color depth represents the fold change, and the circle size represents the number of interacting proteins. Among them, A is the down-regulated protein interaction network analysis in the comparison between the moderate periodontitis group (MoP) and the mild periodontitis group (MiP), and the circles in the figure are down-regulated proteins; B is the up-regulated protein interaction network analysis in the comparison between the severe periodontitis group (SP) and the healthy group (HC), and the circles in the figure are up-regulated proteins. Detailed implementation mode

[0023] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will be described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0024] The following describes the specific implementation of the present invention in detail with specific embodiments.

[0025] Example 1: Purification and identification of salivary exosomes;

[0026] Collect 10 ml of saliva from each volunteer in the healthy group (n = 6), mild (n = 4), moderate (n = 3), and severe (n = 4) periodontitis groups. After diluting with PBS solution at a ratio of 1:1, use differential ultracentrifugation technology. Based on the sedimentation coefficient differences between exosomes and other co-existing contaminants, centrifuge at 800g for 10 minutes to remove cells, take the supernatant and centrifuge at 2000g for 30 minutes to remove dead cells, take the supernatant and centrifuge at 12000g for 45 minutes to remove cell debris, and take the precipitate to sediment exosome particles at a centrifugal force of 100000g to successfully isolate exosomes from saliva.

[0027] 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 in the shape of "tea saucers" or "cups", with diameters ranging from 30 to 150 nm ( Figure 1 as shown in A). The particle size was detected using a nanoparticle sizer. It was found that the particle size of the extracted salivary exosomes showed an obvious single-peak distribution. Most exosomes had particle sizes concentrated between 50 nm and 100 nm, with typical nanoscale size characteristics of exosomes ( Figure 1 as shown in B). At the same time, the proteins of the extracted exosomes were separated by SDS-PAGE electrophoresis using Western Blot technology, and the exosome markers (CD9, CD81, CD63) were identified using specific antibodies ( Figure 1 as shown in C).

[0028] Example 2: Exosome proteomics sequencing and data quality control;

[0029] 1% protease inhibitor was added to the extracted exosome samples, followed by ultrasonic lysis. The protein concentration was measured using a BCA kit. Equal amounts of protein from each sample were digested, and the peptides were desalted using a Strata X SPE column. The polypeptide solution was subjected to chromatographic separation and mass spectrometry identification using an EASY-nLC1200 ultra-high performance liquid system and an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific). Data were collected using a data-independent acquisition (DIA) program. The obtained DIA data were processed using DIA-NN (v.1.8) and the Homo_sapiens_9606_SP_20231220.fasta database, and finally, the identification results and abundances of peptides and proteins were obtained ( Figure 2 as shown in A and B). The principal component analysis (PCA) statistical method was used to evaluate the sample repeatability ( Figure 2 as shown in C). The results showed that the healthy group (HC) and the mild periodontitis group (MiP) were generally separated from the moderate periodontitis group (MoP) and the severe periodontitis group (SP), and the within-group aggregation could reflect the repeatability of the samples.

[0030] Example 3: Screening of differentially expressed proteins;

[0031] Proteins expressed in multiple samples in each group were selected for analysis. A Venn diagram was used to screen for proteins detected in all periodontitis samples ( Figure 3 as shown in A) for subsequent analysis. A heatmap was used to cluster and display the expression levels of the 178 selected proteins. The results showed that the expression patterns of these proteins were divided into 4 types of changing trends that differed with the progression of periodontal disease ( Figure 3In B). To screen for key proteins whose expression levels continuously increase as periodontitis worsens, the ratio of the mean values of the proteins in the two groups of samples was used as the fold change (FC), and a t-test was used to compare the relative quantitative values of the proteins pairwise between groups. A P-value < 0.05 was used to determine the significance of the differences. After logarithmic transformation, a threshold of Log2FC > 0.4 was used to obtain the specific key proteins EHD3 and ATP6V1E1 that continuously increase as periodontitis worsens ( Figure 3 In C). The expression level of EHD3 increased more significantly in mild to moderate periodontitis, and the expression level of ATP6V1E1 increased more significantly in moderate to severe periodontitis, which indicates their main roles at different stages of periodontal disease progression ( Figure 3 In D).

[0032] EHD3 is a membrane-bound protein that is deeply involved in biological processes such as ciliary vesicle formation, endocytic trafficking, and regulation of cell-cell adhesion, and plays an important role in immune dysregulation and cell-cell communication. ATP6V1E1 can acidify and maintain the pH value within organelles. In some biological processes, it can target the plasma membrane, acidify the extracellular environment, and regulate the maturation of osteoclasts.

[0033] Example 4: Protein function and pathway enrichment and clustering analysis;

[0034] 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 discover the enrichment trends of the differentially expressed proteins between groups in terms of functional types and pathways. In the comparison between the mild periodontitis group (MiP) and the healthy control group (HC), activation of the glycolysis pathway and inflammatory response was observed, and at the same time, activation of the non-canonical β-catenin-independent Wnt signaling pathway was enriched, which indicates activation of biological processes such as cell junction, cell migration, and cytoskeleton rearrangement ( Figure 4 ). In the comparison between the moderate periodontitis group (MoP) and the healthy control group (HC), more active immune activation pathways and cell junction regulation pathways were enriched ( Figure 5 ). Activation of these key functions and pathways indicates that in early mild and moderate periodontitis, the high expression of the salivary exosome EHD3 protein regulates cell-cell junctions by activating the non-canonical Wnt pathway, promotes inflammatory infiltration of the periodontal connective tissue and soft tissue destruction, and leads to loss of periodontal tissue attachment.

[0035] In the comparison between the severe periodontitis group (SP) and the healthy control group (HC), multiple functions related to V-type ATPase activation and activation of the insulin signaling (Insulin) pathway were significantly enriched ( Figure 6 and Figure 7). The V-type ATPase family functions as a proton pump, capable of acidifying and maintaining the pH value within organelles, and can also target the cytoplasmic membrane to acidify the extracellular environment, promoting the maturation and differentiation of osteoclasts. Insulin signaling, on the other hand, 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 advanced severe periodontitis, the high expression of the salivary exosome ATP6V1E1 protein can target and activate osteoclast differentiation, while promoting the release of insulin signaling, 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 destruction in severe periodontitis ( Figure 8 ).

[0036] Example 5: Protein-protein interaction network analysis;

[0037] By comparing with the STRING protein-protein interaction network database, the differential protein-protein interaction relationships were extracted according to confidence score > 0.7 (high confidence). In the comparison between the moderate periodontitis group (MoP) and the mild periodontitis group (MiP), the interaction among the downregulated differential proteins EPCAM, KRT18, and SPINT2 was obtained ( Figure 9 in A), which indicates that the epithelial connection and tissue attachment are damaged in the early stage of the periodontal disease process, and the expression of epithelial tissue-related proteins decreases. In the comparison between the severe periodontitis group (SP) and the healthy group (HC), the upregulated differential proteins obtained include the screened key protein ATP6V1E1 and the V-type ATPase family proteins ATP6V0D1, ATP6V0A4, and ATP6V1H ( Figure 9 in B), which further demonstrates the main role of V-type ATPase, especially the key protein ATP6V1E1, in the alveolar bone resorption and destruction in severe periodontitis.

[0038] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. Use of a substance for detecting the expression level of EHD3 protein in salivary exosomes in the preparation of a diagnostic product for early periodontitis.

2. Use of a substance for down-regulating the expression level of EHD3 protein in salivary exosomes in the preparation of a drug for treating, alleviating or improving early periodontitis.

3. Use of a substance for detecting the expression level of ATP6V1E1 protein in salivary exosomes in the preparation of a product for predicting the progression of late periodontitis.

4. Use of a substance for down-regulating the expression level of ATP6V1E1 protein in salivary exosomes in the preparation of a drug for preventing, treating, alleviating or improving late periodontitis.

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