Application of epiinhibitor EED226 in preparation of drug for resisting LPS-induced inflammation of cynoglossus semilaevis

Abstract

The invention relates to application of an epiinhibitor EED226 in preparation of a cynoglossus semilaevis LPS-induced inflammation resisting medicine, and belongs to the field of epigenetics, and the epiinhibitor EED226 reduces LPS-induced inflammation response in a cynoglossus semilaevis body by regulating and controlling expression of downstream immune genes. And the inhibited genes comprise clec4c, ltbr, mcp-1b, tnfrsf13b, tnfsf6 and tnfsf14. The invention further discloses a method for preparing the recombinant protein.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to the application of an epigenetic inhibitor, EED226, in the preparation of an anti-LPS-induced inflammatory drug from half-smooth tongue sole. Background Technology

[0002] Bacterial diseases caused by Gram-negative bacteria pose a serious threat to aquaculture. Gram-negative bacteria can directly damage the health of aquatic animals through toxin secretion, induction of immunosuppression, and various other pathogenic mechanisms, leading not only to significantly increased fish mortality but also to substantial economic losses in the aquaculture industry. As an important component of their outer membrane, lipopolysaccharide (LPS) in mammals can trigger an immune response and induce systemic inflammation by activating the TLR4 signaling pathway. However, although fish lack the typical LPS receptor TLR4 and its co-receptors MD-2 / CD14, they still exhibit a strong inflammatory response when infected with Gram-negative bacteria. Therefore, how the fish immune system recognizes and responds to LPS has become one of the core issues in current fish immunology research. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides the application of the epigenetic inhibitor EED226 in the preparation of an anti-LPS-induced inflammatory drug from half-smooth tongue sole. EED226 can inhibit the expression of downstream immune genes, and its target genes can be mapped. eed The co-expression pattern with downstream immune genes further reveals its mechanism of action.

[0004] The technical solution provided by this invention is as follows: The application of the epigenetic inhibitor EED226 in the preparation of an anti-LPS-induced inflammatory drug for tongue sole. The epigenetic inhibitor EED226 weakens the LPS-induced inflammatory response in tongue sole by regulating the expression of downstream immune genes.

[0005] The application of the epigenetic inhibitor EED226 in the anti-LPS-induced inflammation of tongue sole, wherein the optimal concentration of EED226 is 10 mg and the optimal treatment time is 48 h, and the application is for non-disease treatment or prevention purposes.

[0006] The beneficial effects of this invention compared to existing technologies: This invention demonstrates through research that the epigenetic inhibitor EED226 of the half-smooth tongue sole has an anti-LPS-induced inflammation effect, targeting the gene... eed Co-expression with downstream immune genes. Attached Figure Description

[0007] Figure 1Immunoblot images of spleen cells treated with different concentrations and durations of EED226 are shown. A shows the protein changes of EZH2 after 48 hours of treatment with a combination of LPS (1 mg / ml) and different concentrations of EED226. B shows the protein changes after 48 hours of treatment with a combination of LPS (1 mg / ml) and EED226 (10 mg / ml). + indicates the addition of LPS, and - indicates the absence of LPS.

[0008] Figure 2 This chart shows the comparison, screening, and enrichment analysis of differentially expressed genes in spleen cells of half-smooth tongue sole after LPS and EED226 treatment. A is a volcano plot of differentially expressed genes between LPS and NC; B is a volcano plot of differentially expressed genes between LPS+EED226 and NC; C is a volcano plot of differentially expressed genes between LPS+EED226 and LPS; D is a bar chart of the number of differentially expressed genes in each component; E is a Venn diagram of differentially expressed genes upregulated by LPS vs NC and downregulated by LPS+EED226 vs LPS; F is a bubble chart of KEGG enrichment analysis of the intersection genes between LPS vs NC upregulated and LPS+EED226 downregulated. Figure 3 The diagrams show the identification of downstream pathways and the verification of potential target genes after LPS and EED226 treatment. A is the expression heatmap of genes in the cytokine-cytokine receptor interaction pathway, B is the expression heatmap of genes in the leukocyte transendothelial migration pathway, C and D are the GSEA enrichment analysis of related immune enrichment pathways, and E is the transcriptome expression and fluorescence quantitative detection of downstream potential target genes. Figure 4 This is a diagram illustrating the co-expression pattern of the EED226 target gene and its downstream target genes. A represents... tnfsf14 , mcp-1b , tnfrsf13b At the single-cell level, B is... eed UMAP diagram, C is eed and tnfrsf13b The co-expressed UMAP diagram, D is eed and mcp-1b The co-expressed UMAP diagram, E is eed and tnfsf14 UMAP diagram of co-expression. Detailed Implementation

[0009] The technical solution of the present invention will be further studied through the following embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0010] Application of the epigenetic inhibitor EED226 in the anti-LPS-induced inflammation of half-smooth tongue sole. The optimal concentration of EED226 was 10 mg / mL. The optimal treatment time for EED226 was 48 h. The co-expression pattern was used to map its target genes. eed Single-cell co-expression levels with downstream immune genes.

[0011] The epigenetic inhibitor EED226 was a commercially available formulation (manufacturer: Selleck, catalog number: S8496). Example

[0012] A method for determining treatment concentration and time includes the following steps: 1. Experimental sample: Half smooth tongue sole, about 1 year old, about 36cm in length, and free from disease, were purchased from Taitung Market.

[0013] 2. Primary cell preparation: Spleen tissue from anesthetized and dissected fish was transferred to six-well plates. After removing the surrounding fascia tissue with PBS, the cells were cut into small pieces of 1–2 mm³ using sterile scissors and incubated at 24°C for 30 minutes to promote digestion. After digestion, the cell suspension was passed sequentially through 100 µm and 70 µm cell sieves (Corning, 352360, USA) and collected into 50 ml centrifuge tubes.

[0014] 3. Experimental Design: The dissociated 10 7 Primary cells of *Cyclophorus semismoothienos* were seeded in T25 cell culture flasks to a final volume of 4 ml. Treatment groups were as follows: at 48 h, the control group received only 40 μL of DMSO, the positive control group received 40 µL of LPS (1 mg / ml), and the experimental groups received EED226 (based on extensive literature review) at concentrations of 5 mg and 10 mg / ml. Additionally, the experimental groups received either 1 mg / ml LPS or no LPS at all. Specific group details are as follows: Figure 1 As shown in Figure A. At the remaining 24h and 72h, treatment was performed using a combination of 10 mm EED226 and 1 mg / ml LPS, respectively. After the corresponding time points, RIPA lysis was used to extract total protein for immunoblotting.

[0015] 4. Immunoblotting: After complete lysis, cells were centrifuged at 12,000 × g for 10 minutes, and the supernatant was carefully aspirated. Protein concentration was determined using the BCA protein quantification kit according to the manufacturer's instructions. Subsequently, protein concentration was measured using a TECAN microplate reader. After determining the concentration, 5× sample buffer was added to the supernatant, and the mixture was heated at 95°C until complete denaturation. Based on the determined concentration, an appropriate amount of protein sample was loaded for SDS-PAGE electrophoresis, initially at 80 V for 20 minutes, then increased to 120 V for 1 hour. The PVDF membrane was pre-activated with methanol, and the transfer equipment was prepared in the following order: filter paper, PVDF membrane, protein gel, and filter paper. The gel was immersed in transfer buffer, i.e., Trans-Blot Turbo 5x transfer buffer, and then the gel was placed into the Trans-Blot Turbo 5x transfer buffer. ® In the transfer tank of the semi-dry transfer system, transfer was performed at a constant current of 25 V and 2 A for 10 minutes. After transfer, the membrane was blocked with 5% skim milk powder for 1 hour. Rabbit anti-EED antibody and rabbit anti-actin antibody were diluted according to the recommended ratio and incubated at room temperature for 2 hours. After incubation, the membrane was washed three times with 1× TBST for 5 minutes each time. Subsequently, the membrane was incubated with goat anti-rabbit secondary antibody diluted 1:20,000 at room temperature for 1 hour. After incubation, the membrane was washed three more times with 1× TBST for 10 minutes each time. Then, a 1:1 mixture of ultrasensitive substrate was used for color development. Since the EZH2 content in the LPS+EED226 group did not decrease significantly compared to the LPS group after 24 hours of EED226 treatment, relevant images were not retained, such as... Figure 1 As shown in Figure B, after 48 hours of EED226 treatment, the EZH2 content in the LPS-infected group significantly increased compared to the NC group. However, after the addition of EED226, the EZH2 protein content significantly decreased, accompanied by an increase in EED226 concentration. A treatment concentration of 10 mg / L was the most effective in inhibiting EZH2, representing the optimal concentration. After 72 hours of treatment, the EZH2 expression level in the LPS-treated group did not change significantly compared to the NC group; therefore, 48 hours was the optimal treatment time. Example

[0016] To verify the effect of the epigenetic inhibitor EED226 on LPS-induced inflammation in tongue sole, it was tested in spleen cells of tongue sole: 1. Experimental Design: 10 samples were dissected following the same steps in Example 1. 7Primary cells of *C. semi-smooth tongue sole* were seeded in T25 cell culture flasks. Treatment groups were as follows: the control group (NC) received 40 µl DMSO; the LPS group received 40 µl LPS (1 mg / ml); and the LPS + EED226 group received both 40 µl EED226 (final concentration 10 mg / ml) and 40 µl LPS. The final volume of each group was adjusted to 4 mL, and four biological replicates were set up for each group. After incubation at 24°C for 48 hours, the cells were collected into 1.5 mL centrifuge tubes, trizol was added, and the cells were temporarily stored at -80°C.

[0017] 2. Transcriptome sequencing: Total RNA was extracted following the Trizol procedure. The purity, concentration, and integrity of the extracted total RNA were assessed. Subsequently, mRNA was extracted from the total RNA using magnetic beads containing poly-T oligonucleotides, and a sequencing library was constructed. Sequencing was then performed on the Illumina HiSeq 2000 platform.

[0018] 3. Data Analysis: Transcriptome data were correlated with that of the half-smooth tongue sole (Caulis Half-smooth). Cynoglossus semilaevis The genome (NCBI ID: 24447) was analyzed. Differentially expressed genes were screened and gene enrichment analysis was performed, and the data was visualized. Results are as follows: Figure 2 The number of differentially downregulated genes after LPS infection and EED226 treatment was significantly increased compared to LPS infection. We enriched differentially upregulated genes related to LPS activation and found that these genes were enriched in immune-related pathways such as leukocyte transendothelial analysis, MAPK signaling pathway, and antigen processing and presentation.

[0019] Example 3: A method for screening downstream target genes of EED226, the steps of which are as follows: 1. Extract total RNA following the trizol procedure.

[0020] 2. The purity of the extracted total RNA was determined using a Kaiao K5500® spectrophotometer (Kaiao, Beijing, China). RNA concentration and integrity were assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, California, USA) and an RNA Nano 6000 Assay Kit.

[0021] 3. Subsequently, mRNA was extracted from total RNA using magnetic beads containing poly-T oligonucleotides. Following the kit instructions, 2 μg of total RNA was used to construct sequencing libraries using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA), and appropriate index tags were added to each sample. The libraries were then sequenced on the Illumina HiSeq 2000 platform.

[0022] 4. Sequencing data were aligned to *Sinonovacula semismoothina* using Hisat2 software (version 2.1.1). Cynoglossus semilaevis The genome (NCBI ID: 24447) was analyzed. Differentially expressed genes between the two groups were screened using DESeq (Bioconductor, version 1.14.0). Gene enrichment analysis was performed using DAVID BioinformaticsResources, and data visualization was conducted using the Lianchuan Cloud Platform.

[0023] 5. The expression levels of downstream target genes were determined using quantitative real-time PCR. The results are as follows: Figure 3 As shown, clec4c , ltbr , mcp-1b , tnfrsf13b , tnfsf6 and tnfsf14 Six immune-related genes were significantly downregulated after the addition of EED226 compared to the LPS group.

[0024] Example 3: A method for mapping its target genes eed The method for co-expressing downstream immune genes involves the following steps: 1. The single-cell transcriptome (scRNA-seq) data of half-smooth tongue sole used in this study was downloaded from the Genome Sequence Archive (GSA: CRA017585, https: / / ngdc.cncb.ac.cn / gsa / search?searchTerm=CRA017585) of the National Genome Science Data Center (China National Center for Biotechnology Information / Beijing Institute of Genomics, Chinese Academy of Sciences).

[0025] 2. Both co-expression UMAP plots and single-gene expression UMAP plots are drawn using the FeaturePlot function in the Seurat R package. The former requires setting the parameter "blend=TRUE". Co-expression mode: such as... Figure 4 As shown, eed With downstream immune genes tnfrsf13b, mcp-1b, tnfsf14Co-expression also exists at the single-cell level. tnfrsf13b and eed It is mainly co-expressed in B cells. mcp-1b and eed Mainly co-expressed on T cells and tnfsf14 and eed It is primarily co-expressed on M2 macrophages and T cells. This further illustrates that EED226 plays a regulatory role in downstream immune genes.

Claims

1. The application of the epigenetic inhibitor EED226 in the preparation of an anti-LPS-induced inflammatory drug from half-smooth tongue sole, characterized in that, The epigenetic inhibitor EED226 attenuates LPS-induced inflammatory responses in half-smooth tongue sole by regulating the expression of downstream immune genes.

2. The application of the epigenetic inhibitor EED226 in the anti-LPS-induced inflammation of half-smooth tongue sole, wherein the application is for non-disease treatment or prevention purposes, characterized in that... The optimal treatment concentration of EED226 is 10 mg, and the treatment time is 48 h. The epigenetic inhibitor EED226 reduces the LPS-induced inflammatory response in half-smooth tongue sole by regulating the expression of downstream immune genes.

3. The application according to claim 1 or 2, characterized in that, Suppressed immune genes include clec4c , ltbr , mcp-1b , tnfrsf13b , tnfsf6 and tnfsf14 .

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