Application of zebrafish 80S ribosomal protein 7oyb in influence of DON on inhibition of zebrafish embryo fibroblast proliferation
By studying the binding site of zebrafish 80S ribosomal protein 7oyb, it interferes with the binding of the vomiting toxin DON and zebrafish ribosomal protein 7oyb, the problem of DON inhibiting the proliferation of zebrafish embryonic fibroblasts was solved, revealing its cytotoxic mechanism, and achieving effective interference with cell proliferation.
Patent Information
- Application Number
- CN202510580147.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-08
AI Technical Summary
The mechanism of action of vomiting toxin DON in aquatic model biological cells in the prior art is unclear, and the specific mechanism affecting cell proliferation has not been studied in depth, making it difficult to effectively respond to food safety and health threats.
By studying the binding site of zebrafish 80S ribosomal protein 7oyb, it interferes with the binding of the vomiting toxin DON and zebrafish ribosomal protein 7oyb, specifically through hydrogen bonding and hydrophobic interaction, it interferes with its binding to 18s RNA and 40S ribosomal protein S19, affecting its proliferation of zebrafish embryonic fibroblasts.
For the first time, the site of action that DON inhibits the proliferation of zebrafish embryonic fibroblasts was clarified. By interfering with the binding of these sites to DON, the problem of inhibiting cell proliferation by DON was solved and its cytotoxic mechanism was revealed.
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Figure CN120441675A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to an application of zebrafish 80S ribosomal protein 7oyb in influencing DON to inhibit the proliferation of zebrafish embryonic fibroblasts. Background Art
[0002] Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced by Fusarium spp. and widely found in contaminated grains and their products. DON is a chemically and thermally stable compound that rarely decomposes during storage or heat treatment, leading to its widespread presence in foods consumed by humans and animals. DON has multiple toxic effects on humans and animals, including immunotoxicity, reproductive toxicity, and neurotoxicity, posing a serious threat to food safety and human health.
[0003] Cell proliferation is a fundamental process for biological growth, development, and tissue homeostasis. Understanding the mechanisms by which DON affects cell proliferation is crucial for understanding its toxicity, developing detoxification strategies, and ensuring food safety. While current research focuses on the effects of DON on mammalian cells, its mechanisms of action in aquatic model organisms remain largely undefined. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a use of zebrafish ribosomal protein 7oyb in influencing DON to inhibit the proliferation of zebrafish embryonic fibroblasts. The present invention uses methods such as RNA-seq, GSEA, molecular docking, cell immunofluorescence, flow cytometry, and confocal microscopy to clarify the mechanism by which DON binds to the ribosomes of zebrafish embryonic fibroblasts, leading to reduced DNA replication, cell cycle arrest, and interference with mitosis, ultimately affecting cell proliferation.
[0005] The present invention studies the specific action pathway of DON on the proliferation of zebrafish embryonic fibroblasts and finds that DON produces cytotoxicity by binding to the amino acid site on the zebrafish ribosomal protein 7oyb, thereby inhibiting the proliferation of zebrafish embryonic fibroblasts.
[0006] The present invention is achieved through the following technical solutions:
[0007] The invention relates to the use of zebrafish 80S ribosomal protein 7oyb in influencing vomitoxin to inhibit the proliferation of zebrafish embryonic fibroblasts. The application involves interfering with the binding site of vomitoxin on the zebrafish 80S ribosomal protein 7oyb by affecting the binding site of vomitoxin on the zebrafish 80S ribosomal protein 7oyb. The specific binding is as follows: vomitoxin forms hydrogen bonds with nucleotides G17192, C17202, and C17312 on 18S RNA, and forms hydrophobic interactions with G15932 on 18S RNA and PRO90 and HIS92 of 40S ribosomal protein S19. G17192, C17202, C17312, and G15932 are positions 1717, 1718, 1730, and 1593 on SEQ ID NO. 1, respectively, and PRO90 and HIS92 are amino acids 90 and 92 on SEQ ID NO. 2, respectively.
[0008] The present invention also provides the use of vomitoxin in inhibiting the proliferation of zebrafish embryonic fibroblasts. The application method comprises the following steps: vomitoxin binds to zebrafish ribosomal protein 7oyb, thereby inhibiting the proliferation of zebrafish embryonic fibroblasts. The specific binding is as follows: vomitoxin forms hydrogen bonds with amino acids G17192, C17202, and C17312 on 18s RNA, and forms hydrophobic interactions with G15932 on 18s RNA and PRO90 and HIS92 of 40S ribosomal protein S19. G17192, C17202, C17312, and G15932 are positions 1717, 1718, 1730, and 1593 on SEQ ID NO. 1, respectively; PRO90 and HIS92 are amino acids 90 and 92 on SEQ ID NO. 2, respectively.
[0009] The present invention has the following beneficial effects compared with the prior art: The present invention is the first to discover the sites of action of DON in inhibiting the proliferation of zebrafish embryonic fibroblasts. By interfering with the binding of these sites with DON, the problem of DON inhibiting the proliferation of zebrafish embryonic fibroblasts can be solved. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 DON inhibits the proliferation of zebrafish embryonic fibroblasts; A shows the effect of different concentrations of DON on zebrafish embryonic fibroblasts for 48 hours on cell viability; B shows the effect of different concentrations of DON on zebrafish embryonic fibroblasts for 96 hours on cell viability; C shows the changes in the inhibition of zebrafish embryonic fibroblast proliferation by different concentrations of DON over time; D shows the effect of different concentrations of DON on the proliferation of zebrafish embryonic fibroblasts using confocal microscopy, where the images from left to right are 0 ng / mL, 600 ng / mL, and 1000 ng / mL;
[0011] Figure 2 DON treatment leads to down-regulated pathways and related genes; A is the ribosome pathway KO03010 and down-regulated gene heat map; B is the DNA replication pathway KO03030 and down-regulated gene heat map; C is the cell cycle KO04110 and down-regulated gene heat map; D is the ECM-receptor action KO04512 and down-regulated gene heat map;
[0012] Figure 3 DON and zebrafish ribosomal 80s protein 7oyb molecular docking; A is a bar graph of ribosomal pathway downregulated gene expression; B is a 2D image of DON and zebrafish ribosomal 80s protein 7oyb molecular docking; C is a 3D image of DON and zebrafish ribosomal 80s protein 7oyb molecular docking;
[0013] Figure 4 DON induces cell cycle arrest and mitotic disorders; A is the relative expression of cell cycle-related genes verified by qPCR; B is the percentage of each cell cycle stage; C is a flow cytometry analysis of the cell cycle; D is a confocal micrograph of mitotic disorders, with DAPI dye labeling the nucleus and aTubulin antibody labeling the spindle; E is a statistical histogram of multinucleated cells; F is a statistical histogram of mitotic disorders;
[0014] Figure 5 DON leads to decreased levels of extracellular matrix and fibronectin; A is qPCR verification of the expression levels of key genes in the extracellular matrix and fibronectin pathways; B is confocal microscopy analysis of changes in fibronectin, with DAPI dye labeling cell nuclei and Fibronectin labeling fibronectin. DETAILED DESCRIPTION
[0015] The technical solution of the present invention is further explained below through examples, but the protection scope of the present invention is not limited in any form by the examples.
[0016] Example 1
[0017] (1) Cell proliferation: CCK-8 and EdU detection kits were used to analyze the effect of DON on cell proliferation.
[0018] Zebrafish embryonic fibroblasts were seeded in a culture dish. When the cells adhered to the wall and grew to 60%-70% confluence, different concentrations of DON (DON 0ng / mL, 200ng / mL, 400ng / mL, 600ng / mL, 800ng / mL, 1000ng / mL, 1200ng / mL) were added to treat the cells. At different time points (48h and 96h), DON 0ng / mL was set as a control. CCK-8 reagent was used and the effect of DON treatment on cell viability was detected by a microplate reader at 450nm. The results showed that DON could inhibit the proliferation of zebrafish embryonic fibroblasts at both 48h and 96h ( Figure 1 A and B in Figure 3), and when the DON concentration was 600 ng / mL, cell proliferation decreased significantly from 0 to 96 h, and cell proliferation decreased more at DON1000 ng / mL than at DON600 ng / mL, indicating that DON inhibited cell proliferation in a concentration-dependent manner ( Figure 1 C).
[0019] The cells were plated on a slide and treated with DON (0 ng / mL, 600 ng / mL, 1000 ng / mL) for 48 h. EdU reagent was then added and incubated for 2 h to label the cells. The cells were then fixed with 4% paraformaldehyde at room temperature for 15 minutes and permeabilized with 0.3% Triton X-100 at room temperature for 15 minutes. The reaction solution was prepared for EdU staining (488). The staining conditions were incubated at room temperature for 30 minutes. The nuclei were then stained with DAPI. The staining conditions were incubated at room temperature for 10 minutes. Confocal microscopy was used to analyze the effect of DON on the proliferation of zebrafish embryonic fibroblasts. The results showed that DON inhibited cell proliferation. As the concentration of DON increased, cell growth slowed significantly ( Figure 1 D) in.
[0020] (2) Transcriptome analysis was used to analyze the transcriptional regulatory network after DON action to reveal the complex regulatory mechanism of DON on cell proliferation.
[0021] Zebrafish embryonic fibroblasts were plated on culture dishes and treated with various concentrations of DON (0 ng / mL, 600 ng / mL, and 1000 ng / mL) for 48 hours. The cells were then harvested and RNA extracted. Poly(A)-tailed eukaryotic mRNA was enriched using magnetic beads with oligo(dT) and then ultrasonically fragmented. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first-strand cDNA was synthesized using the M-MuLV reverse transcriptase system. The RNA was then degraded with RNase H, and the second-strand cDNA was synthesized using dNTPs in the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and ligated with sequencing adapters. AMPure XP beads were used to screen cDNA of approximately 200 bp, followed by PCR amplification. PCR products were purified again using AMPure XP beads to construct libraries. Agarose gel electrophoresis was then used to analyze the integrity of the sample RNA and the presence of DNA contamination. A NanoPhotometer spectrophotometer was used to detect RNA purity (OD260 / 280 and OD260 / 230 ratios). A Qubit2.0 Fluorometer was used to accurately quantify RNA concentration. An Agilent 2100 bioanalyzer was used to accurately detect RNA integrity, followed by sequencing analysis.
[0022] RNA-seq, GSEA analysis and gene heatmap were combined to analyze the significantly downregulated and upregulated pathways and analyze the significantly differentially expressed genes. It was found that after DON treatment of cells, the ribosome pathway, DNA replication pathway, ECM-receptor pathway and cell cycle pathway were significantly downregulated ( Figure 2 A, B, C and D), key genes in the ribosomal pathway, such as those encoding ribosomal proteins (rps9, rps24 and rps28), encoding mitochondrial ribosomal proteins (mrps7), encoding large ribosomal subunit proteins (rpl12 and rpl21) and encoding mitochondrial large ribosomal subunit proteins (mrpl9), were significantly down-regulated ( Figure 2 China A and Figure 3 Figure 3 (A), indicating that DON exposure affects ribosomes in zebrafish embryonic fibroblasts.
[0023] (3) Molecular docking analysis was used to determine whether DON binds to zebrafish ribosomes.
[0024] To further analyze the interaction between DON and the ribosome, molecular docking was used to analyze the interaction between DON and the zebrafish 80S ribosome. We used the zebrafish 80S ribosomal protein (PDB: 7oyb). The CAS number for DON is 51481-10-8. First, we preprocessed DON and the protein using AutoDockTools software (Version 1.5.7), selected appropriate docking sites, and exported a configuration file for docking with AutoDockVina software. Next, we used AutoDockVina software to perform local searches and repeated iterations to find the optimal molecular docking conformation. Finally, we used PyMOL software (Version 3.0.3) and LigPlot software (Version 2.2.9) for analysis and plotting. The binding energy for DON-ribosomal protein docking was -8.4 kcal / mol, indicating that the binding is primarily through hydrogen-bonded hydrophobic interactions. The nucleotides that form hydrogen bonds with DON are G17192, C17202, and C17312 of 18s RNA. The nucleotides that form hydrophobic interactions with DON are G15932 of 18s RNA, PRO90 of 40S ribosomal protein S19, HIS92 ( Figure 3 ).
[0025] (4) Flow cytometry analysis of the effect of DON on cell cycle.
[0026] After DON treatment, cells were added to propidium iodide (PI) staining solution and incubated in the dark. Cytoflex S (Beckman, USA) and CytExpert software were used for detection and data acquisition. Flowjo software was used to analyze the detection data. The results showed that DON induced cell cycle arrest, resulting in reduced DNA replication. This effect was significantly positively correlated with DON concentration ( Figure 4 ).
[0027] (5) Immunofluorescence and confocal microscopy were used to analyze the effects of DON on mitosis and extracellular matrix.
[0028] The cells were plated and treated with DON for 48 hours. The cells were fixed with 4% PFA, permeabilized with 0.4% TritonX-100, and blocked with blocking solution. After incubation with primary antibodies (Fibronectin, α-tubulin), the corresponding fluorescent secondary antibodies were incubated, and the cell nuclei were labeled with DAPI. Laser confocal microscopy was performed. The results showed that after DON stimulation, the number of multinucleated cells increased, cell mitosis was impaired, and DNA distribution was abnormal ( Figure 4 ), fibronectin appears as dots ( Figure 5 ).
[0029] 7OYB_1|Chain A[auth 22]|18S rRNA|Zebrafish (7955) (the bold underlined black markers indicate sites G15932 / G17192 / C17202 / C17312):
[0030]
[0031]
[0032]
[0033] >7OYB_42|Chain PA[auth T2]|40S ribosomal protein S19|Zebrafish (7955) (the bold and underlined areas are PRO90 and HIS92 sites):
[0034]
Claims
1. Application of zebrafish 80S ribosomal protein 7oyb in influencing vomitoxin to inhibit the proliferation of zebrafish embryonic fibroblasts, characterized in that: The application is to interfere with the binding of vomitoxin to zebrafish 80S ribosomal protein 7oyb by affecting the binding site of vomitoxin on zebrafish 80S ribosomal protein 7oyb. The specific binding is as follows: vomitoxin forms hydrogen bonds with nucleotides G17192, C17202, and C17312 on 18S RNA, and forms hydrophobic interactions with G15932 on 18S RNA and PRO90 and HIS92 of 40S ribosomal protein S19. The G17192, C17202, C17312, and G15932 are positions 1717, 1718, 1730, and 1593 on SEQ ID NO. 1, respectively, and PRO90 and HIS92 are amino acids 90 and 92 on SEQ ID NO. 2, respectively.
2. The use of vomitoxin in inhibiting the proliferation of zebrafish embryonic fibroblasts, characterized in that: The application method comprises the following steps: combining vomitoxin with zebrafish ribosomal protein 7oyb to inhibit the proliferation of zebrafish embryonic fibroblasts. Specifically, the vomitoxin forms hydrogen bonds with amino acids G17192, C17202, and C17312 on 18s RNA, and forms hydrophobic interactions with G15932 on 18s RNA and PRO90 and HIS92 of 40S ribosomal protein S19. G17192, C17202, C17312, and G15932 are positions 1717, 1718, 1730, and 1593 on SEQ ID NO. 1, respectively; and PRO90 and HIS92 are amino acids 90 and 92 on SEQ ID NO. 2, respectively.