Application of leucine repeat protein 8D in prevention and treatment of silicosis
By studying the expression and function of leucine repeat protein 8D (LRRC8D), drugs used to inhibit silicosis fibrosis are prepared, which solves the problem of lack of effective diagnostic and therapeutic measures in the prior art, provides new therapeutic targets and diagnostic methods, and delays the process of silicosis fibrosis.
Patent Information
- Application Number
- CN202510096452.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art lacks effective early screening diagnostic methods and special therapeutic measures to deal with pulmonary fibrosis caused by silicosis, and lacks pulmonary fibrosis process evaluation indicators and clear therapeutic targets.
Leucine repeat protein 8D (LRRC8D) was used as a drug component to study its expression and function in silicosis through single-cell RNA sequencing and spatial transcriptome sequencing technology, and drugs used to inhibit silicosis fibrosis were prepared.
LRRC8D is upregulated in silicosis, and is involved in the activation of macrophages and the migration of fibroblasts, affecting the process of pulmonary fibrosis, providing new therapeutic targets and diagnostic methods, and delaying the process of silicosis fibrosis.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of leucine repeat protein 8D in preventing and treating silicosis fibrosis. Background Art
[0002] Silicosis is a chronic occupational disease caused by inhaled silicon dioxide (SiO2) and characterized by progressive, diffuse pulmonary fibrosis. Once it occurs, it will develop progressively, which will not only endanger the health of workers exposed to silica dust, reduce their working ability and quality of life, but also cause huge economic losses to the country. At the same time, silicosis is a global problem and is more common in low- and medium-developed countries. Since little is known about the pathogenesis of silicosis, clinical diagnosis and treatment mainly face two problems: (1) There is a lack of screening and diagnostic methods in the early stage. When the diagnosis is confirmed by chest X-ray, the lung damage cannot be reversed; (2) There is a lack of specific treatment measures for late pulmonary fibrosis. This is because there is currently a lack of evaluation indicators for the progression of pulmonary fibrosis and there is no clear treatment target in clinical practice. Alveolar macrophages (AMO) are the main effector cells of silicosis. Macrophage activation is the starting point of silicosis and plays an important role in the occurrence and development of silicosis. First, alveolar macrophages gather and phagocytize silica dust that enters the alveoli, releasing silica dust particles and cell contents, causing damage and death of a large number of dust cells; in addition, dead dust cells release bioactive substances such as reactive oxygen and cytokines, leading to damage to alveolar structure and alveolar epithelial cells, and ultimately leading to pulmonary tissue fibrosis. Therefore, in the early stage of pulmonary fibrosis caused by silica dust, pulmonary macrophages play a very important role.
[0003] Leucine-rich repeat-containing 8 D (LRRC8D) is the fourth member of the leucine repeat protein family VIII. Currently, it is mainly involved in the occurrence and development of blood diseases and diabetes, and its role in inflammatory response and fibrosis development in silicosis has not been reported.
[0004] LRRC8D is a bulk anion channel protein with a molecular weight of about 120 kDa. It plays an ion channel function in cell transmembrane transport. Although studies have shown that LRRC8D acts as a receptor for lymphocyte development and adipocyte differentiation, the mechanism of its biological effects is still unclear due to its isomeric specificity. It is currently mainly involved in the transport of small molecules, such as the antibiotic blastocystin S7, chemotherapy drugs platinum (cisplatin and carboplatin), and neurotransmitters glutamate, aspartate, and γ-aminobutyric acid. At present, with the rise of single cell RNA sequencing (scRNA-seq) and spatial transcriptome sequencing technology, lung diseases can be studied in depth. Single cell sequencing and spatial transcriptomics technology reveal changes in cell spatial structure through the form of gene four-dimensional spatial expression, providing a new direction for in-depth exploration of cell fate and mechanism research, and providing new opportunities for further exploration of LRRC8D's biological effects in the process of silicosis fibrosis. Summary of the invention
[0005] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:
[0006] Application of LRRC8D in the preparation of medicines for treating silicosis.
[0007] Furthermore, the treatment of silicosis refers to inhibiting fibrosis of silicosis.
[0008] Further, the silicosis is silica-induced.
[0009] Furthermore, the SiO2 promoted the increase of LRRC8D in macrophages.
[0010] A drug for treating silicosis, comprising LRRC8D.
[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.
[0012] Furthermore, the dosage form of the drug includes tablets, granules, capsules, powders or injections.
[0013] Application of LRRC8D in the preparation and improvement of drugs for silicotic fibrosis.
[0014] Further, the silicotic fibrosis is silica-induced.
[0015] Beneficial effects of the present invention:
[0016] 1. The present invention discloses the complete gene information of LRRC8D in the temporal and spatial dimensions of SiO2-induced silicosis AMO upregulation, as well as the regions of AMO cell aggregation distribution in a mouse-induced silicosis fibrosis model;
[0017] 2. The present invention discloses that LRRC8D is expressed in silicosis patients and upregulated in mouse AMO;
[0018] 3. The present invention discloses that LRRC8D participates in the biological function of AMO-induced fibroblast migration in the process of silicosis fibrosis;
[0019] 4. The present invention discloses that LRRC8D participates in SiO2-promoted AMO polarization and is involved in the biological functions related to M2-type pulmonary fibrosis;
[0020] 5. The present invention discloses that LRRC8D resides in the endoplasmic reticulum and functions as a non-ion channel to promote pulmonary fibrosis;
[0021] 6. LRRC8D provides a new therapeutic target for the diagnosis and treatment of silicosis and for delaying silicosis fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0023] Figure 1 A is the present invention based on single cell RNA sequencing and spatial transcriptome sequencing technology to explore the expression of SiO2 in alveolar macrophage LRRC8D gene, Figure 1 B is the spatial transcriptome sequencing technology of the present invention to explore the LRRC8D aggregation and distribution area;
[0024] Figure 2 A is HE staining, immunohistochemical staining and protein quantitative analysis experiments of the present invention revealing the expression of LRRC8D protein in clinical patients with silicosis; Figure 2 BC is expressed by mouse AMO cells of the present invention;
[0025] Figure 3 The present invention uses LRRC8D small molecule interfering RNA (siRNA) technology to knock out the target protein in AMO, collects AMO conditioned medium to stimulate fibroblasts, and uses cell migration experiments to explore the biological behavior of LRRC8D involved in SiO2-induced fibroblast migration;
[0026] Figure 4 The present invention shows that LRRC8D is involved in SiO2-induced macrophage polarization: GO functional enrichment and KEGG signaling pathway analysis are performed in combination with macrophage DEGs in single-cell data to explore the phenotypic changes of AMO polarization promoted by SiO2, and the biological effects caused by LRRC8D participating in AMO polarization;
[0027] Figure 5 A is the SiO2 of the present invention that induces LRRC8D to reside in the endoplasmic reticulum and accelerates pulmonary fibrosis: a chloride ion kit is used to detect changes in chloride ion concentration. Figure 5 B is the molecular mechanism of the protein quantitative experiment of the present invention to detect the molecular translocation phenomenon of LRRC8D residing in the endoplasmic reticulum non-cellular membrane. DETAILED DESCRIPTION
[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] Experiment 1 Single-cell RNA sequencing ( Figure 1 A) and spatial transcriptome ( Figure 1 B) Technical detection of LRRC8D gene expression and spatial distribution in macrophages:
[0030] Male C57BL / mice (5-6 weeks old, 22-30g) were randomly divided into two groups: normal saline (NS) group for 56 days and SiO2 group for 56 days. One high-quality mouse was selected for each group. Silicosis model was established by tracheal instillation of SiO2 suspension (0.2g·kg-1, 50mg·mL-1). The control group mice were given the same volume of NS. On the 56th day, the right lung was removed for single-cell sequencing and the left lung for spatial transcriptome sequencing. Principal component analysis technology, uniform manifold approximation and projection dimensionality reduction were used to capture cell clustering, and spatial transcriptome sequencing technology was used to explore the original position of the LRRC8D gene on lung tissue sections and its mapping in AMO.
[0031] Combination Figure 1 The data in the table show that single-cell sequencing results suggest that LRRC8D is enriched in silicosis AMO ( Figure 1 Spatial transcriptome sequencing further showed that these enriched AMOs were concentrated in the inflammatory regions of SiO2-induced silicosis ( Figure 1 B). Conclusion: Single-cell sequencing and spatial transcriptome sequencing methods were used to detect that LRRC8D was significantly enriched in AMO and inflammatory areas at the transcriptional level in SiO2-induced silicosis animal models, and may be involved in the inflammatory response of silicosis.
[0032] Experiment 2: SiO2 promotes the increase of LRRC8D in clinical samples and mouse macrophages:
[0033] Clinical silicosis patient (male, 57 years old) was stained with hematoxylin-eosin (HE) and showed obvious silicosis nodules ( Figure 2 A), compared with healthy people; immunohistochemical staining showed that LRRC8D was mainly expressed in the interstitial tissue of lung ( Figure 2 In addition, mouse macrophages RAW264.7 were cultured at 1×10 5 The cells were inoculated in a 24-well plate and cultured under 5% CO2 and 37°C. When the cell adhesion reached 85%, 50 μg·cm-2 SiO2 suspension was given to stimulate the cells at 0, 1, 3, 6, 12 and 24 h, respectively. Three replicates were performed at each time point. The cells were collected and treated with cell lysate for protein quantitative analysis of LRRC8D expression (B). SPSS21.0 statistical software was used to analyze the experimental results (C, **P<0.01). Figure 2 It can be seen that HE staining can show obvious silicosis nodules in patients with silicosis ( Figure 2 Middle A, upper right), healthier people ( Figure 2 Immunohistochemical staining showed that LRRC8D was widely distributed in the interstitial area of silicosis patients ( Figure 2 Middle A (lower right picture), healthier people ( Figure 2 At the same time, Western Blot method detected the expression of LRRC8D in mouse macrophages (RAW264.7) in vitro, which increased in a time-dependent manner. The most obvious increase was 6-12 hours after SiO2 treatment ( Figure 2 Middle B), **P<0.01, *P<0.05 ( Figure 2 Conclusion: SiO2 induces the upregulation of LRRC8D expression in lung interstitial cells of clinical silicosis patients and in vitro mouse macrophages.
[0034] Experiment 3: Knockout of LRRC8D reversed the migration of fibroblasts induced by inflammatory mediators released by SiO2-induced macrophage activation:
[0035] The cell migration ability was determined by wound healing assay. MLG (mouse fibroblasts) were cultured at 1×10 5 The cells were seeded in a 24-well plate at a density of 10 cells and incubated at 37°C until the cells were 90% confluent. A 200 μl sterile pipette was then used to draw a medium-width line vertically in the middle of the 24-well plate. Washed three times with PBS, and then the supernatant of new RAW264.7 with LRRC8D knockout was added to each well and co-incubated with MLG. Digital photos of the scratch gap were obtained at 0, 6, 12, and 24 hours, of which 12 hours reversed the migration ability of fibroblasts. The distance between the cell gaps was quantified using ImageJ. Figure 3 It can be seen that: LRRC8D was knocked out in alveolar macrophages by siRNA knockout technology, and the macrophage supernatant was collected to stimulate mouse lung fibroblasts for 12 days. The 2D migration results showed that compared with the control group, knocking out LRRC8D significantly inhibited the migration of mouse lung fibroblasts induced by SiO2. Conclusion: Knocking out LRRC8D inhibits the migration of lung fibroblasts induced by SiO2, suggesting that LRRC8D is involved in the process of silicosis fibrosis.
[0036] The sequence of the siRNA is: CTCAGATGCTTAGACGTCATT.
[0037] Experiment 4 LRRC8D is involved in SiO2-induced macrophage polarization:
[0038] Metascape online website (https: / / metascape.org / gp / index.html# / main / step1) was used to perform GO functional enrichment and KEGG signaling pathway analysis on macrophage DEGs, and the biological processes, molecular functions, cellular components, and signaling pathways involved in DEGs were analyzed. The inclusion criteria were the top 5 enriched numbers of genes in each part and P < 0.05. Figure 4 It can be seen that the gene function enrichment and related signal pathway analysis of the macrophage gene database using single-cell sequencing suggest that SiO2 induces macrophage polarization (M1, M2 type), participates in the migration, adhesion and cell regulation of M1 macrophages, participates in the development regulation, cell adhesion and migration of M2 macrophages, and the proliferation and differentiation of fibroblasts. Conclusion: LRRC8D participates in SiO2-induced macrophage polarization and affects the process of silicosis fibrosis.
[0039] Experiment 5: SiO2 induces LRRC8D to reside in the endoplasmic reticulum and accelerates pulmonary fibrosis:
[0040] Chloride ion concentration determination: RAW 294.7 cells stimulated with SiO2 were collected at 0.5, 1, 3, 6, and 12 hours, and the supernatant was obtained by centrifugation. A total volume of 50ul suspension (5ul reagent + 45ul double distilled water) was prepared, and 200ul was added to each space. The mixture was mixed thoroughly and allowed to stand at room temperature for 15 minutes. A standard curve was prepared and the samples were detected at a wavelength of 620nm.
[0041] Plasma membrane and endoplasmic reticulum protein extraction: Separate plasma membrane and endoplasmic reticulum proteins from RAW 264.7 cells, collect cells, wash repeatedly with ice PBS 2-3 times, add protease inhibitors to the cell pellet, and perform ultrasonic disruption to release endoplasmic reticulum membrane proteins. After disruption, centrifuge at 12000 for 5 minutes to separate the supernatant and precipitate;
[0042] Protein extraction solution treatment: The supernatant was transferred to a centrifuge tube and centrifuged for 10 minutes to separate the endoplasmic reticulum membrane proteins. The precipitate was then treated with protein extraction solution C and ultracentrifuged for 45 minutes to fully lyse the endoplasmic reticulum membrane proteins.
[0043] Membrane protein separation and purification: The cleaved endoplasmic reticulum membrane protein is purified by affinity chromatography. Through adsorption and elution, pure endoplasmic reticulum membrane protein can be obtained. The protein quantitative method is used to detect the levels of LRRC8D, Calnexin and ATP1B1. Figure 5 It can be seen that Cl - Concentration determination suggests that SiO2 has an effect on Cl - The concentration had no effect (A), but increased the expression of LRRC8D and calnexin on the endoplasmic reticulum membrane (B), and had no change on the expression of cell membrane protein ATP1B1. Conclusion: SiO2 induces LRRC8D to reside in the endoplasmic reticulum and accelerates the process of pulmonary fibrosis.
[0044] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0045] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. Application of drugs that inhibit LRRC8D expression in the preparation of drugs for the treatment of silicosis.
2. The use according to claim 1, characterized in that: The treatment involves inhibiting fibrosis in silicosis.
3. The use according to claim 1 or 2, characterized in that: The silicosis referred to herein refers to silica-induced silicosis.
4. The use according to claim 3, characterized in that: The treatment involves inhibiting the elevation of LRRC8D in lung macrophages.
5. A drug for treating silicosis, characterized in that: The medicine contains siRNA knockout reagent.
6. The drug according to claim 5, characterized in that The sequence of the siRNA is SEQ ID NO.
1.
7. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.
8. The drug according to claim 7, characterized in that The dosage form of the drug includes tablets, granules, capsules, powders or injections.
9. Use of a reagent for detecting LRRC8D expression in the preparation of a kit for detecting SiO2-induced silicosis inflammation.