KR20210146765A - Enterocyte-based Quantitative Combination of Proinflammatory Sentinels Specific to 8-keto-Trichothecene - Google Patents

Abstract

The present invention relates to a cell-based bioanalysis system for predicting inflammatory action of 8-keto-trichothecene in intestinal epithelial cells. The cell-based bioanalysis system predicts inflammatory action of 8-keto-trichothecene in human intestinal epithelial cells based on the point that 8-keto-trichothecene induces EGR-1 while suppressing p65 promoter activity in human intestinal epithelial cells. A molecule-based dual biomonitoring system in intestinal epithelial cells according to the present invention can be used as a useful auxiliary tool which reacts with 8-keto-trichothecene in a food matrix to detect a typical molecular inflammation path.

Description

{Enterocyte-based Quantitative Combination of Proinflammatory Sentinels Specific to 8-keto-Trichothecene}

The present invention relates to a cell-based bioanalysis system for predicting the inflammatory action of 8-keto-trichothecene in intestinal epithelial cells, and more specifically, to a tricotine contaminated with crops used as a staple food. It relates to a bio-monitoring system constructed in vitro using intestinal mucosal epithelial cells to detect tessen mycotoxin.

Trichothecenes are a group of more than 200 sesquiterpenoid fungal metabolites, some of which are health-threatening mycotoxins that induce eukaryotic ribosome stalling or cellular ubiquitin depletion. Due to diffuse contamination in the environment and agricultural products such as grains, extensive investigations have been made for the toxic tricotecene. Moreover, some trichothecenes are released from fungal-infected crops into surface soil water and drainage systems. Trichothecenes are characterized by a tetracyclic scirpenol ring system and are classified into four groups (type AD) according to the presence of functional moieties. Field fungi, such as Fusarium species, mainly produce type A and B tricotecenes. Type A trichothecenes are characterized by a hydroxyl or acyl moiety in the R1, R2, R3 or R5 positions of the tetracyclic skiphenol ring. Type B group toxins are commonly named 8-keto-tricotecene due to the ketone group of R5. The ribosome-inactivating activity of trichothecene is related to the presence or absence of substitution of both R1 and R2 with intact 9, 10 double bonds, C-12, 13 epoxides.

Epidemiological studies have confirmed a positive association between human exposure to tricotecene and gastrointestinal disorders, including gastroenteritis and esophageal cancer. In particular, 8-keto-tricotecene, such as deoxynivalenol (DON) and nivalenol (NIV), can cause various mycotoxicities in humans and animals. Acutely poisoned animals show severe damage to the gastrointestinal tract and highly proliferating immune-related cells, resulting in diarrhea, vomiting, gastrointestinal bleeding and leukopenia. In addition to direct tissue damage, DON-exposed mice exhibit cytokine storm syndrome during mucosal and systemic poisoning processes. The epithelial response is the initial gastrointestinal activity that occurs in response to dietary-derived contamination. During mucosal and systemic exposure to trichothecene, the intestinal epithelial lining detects luminal damage and promotes molecular reprogramming. The epithelial barrier responds to luminal damage that activates stress-responsive cellular signals such as mitogen-activated kinases or other pathological biochemical pathways. Then, the activated stress kinase changes the profile of a gene that responds immediately, such as early growth response gene 1 (EGR-1), which through its own pathological state process or regulated gene product, Contributes to maintaining tissue integrity. Moreover, in response to mucosal microbial antigens, pattern recognition receptors are activated and transduce risk signals through key proinflammatory transcription factors including nuclear factor κB (NF-κB). In addition, EGR-1 is involved in a variety of inflammatory cell activities, including inflammatory cell migration and cytokine production.

In spite of various detection methods for trichothecene metabolites, existing analytical techniques are often insufficient to distinguish biological activities in contaminated matrices, and thus more accurate and effective detection methods are needed.

Korean Patent Registration No. 10-1640487 (Registered on July 12, 2016)

Accordingly, the present invention predicts the inflammatory action of 8-keto-trichothecene in intestinal epithelial cells, cell-based 8-keto-trichothecene detection through double reporter analysis An object of the present invention is to provide a system and a cell-based 8-keto-tricotecene detection method using the system.

In order to solve the above problems, the present invention provides a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and a cell-based 8-keto-trichothecene detection system comprising a second reporter recombinant vector comprising a p65 promoter gene and a secreted alkaline phosphatase (SEAP) reporter gene. .

In addition, the present invention provides: 1) a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and transfecting intestinal epithelial cells with a second reporter recombinant vector comprising a p65 promoter gene and a secreted alkaline phosphatase (SEAP) reporter gene; 2) processing the sample to the transfected intestinal epithelial cells; and 3) measuring LUC activity and SEAP activity in intestinal epithelial cells treated with the sample.

The present invention relates to a cell-based bioassay system for predicting the inflammatory action of 8-keto-tricotecene in intestinal epithelial cells, wherein 8-keto-tricotecene inhibits the p65 promoter activity in human intestinal epithelial cells and produces EGR-1 A cell-based bioassay system for predicting the inflammatory action of 8-keto-tricotecene in human intestinal epithelial cells based on induction. The molecular-based dual biomonitoring system in intestinal epithelial cells according to the present invention can be utilized as a useful auxiliary tool to detect typical molecular inflammatory pathways in response to 8-keto-tricotecene in a food matrix.

1 shows the effect of inflammagen on a dual reporter system in human IECs. (A) Schematic representation of the mechanism of a dual reporter system (SEAP for p65 transcription and luciferase for EGR-mediated transcription) for detecting 8-keto-tricotecene in PSMA3-deficient IECs. (BD) Shows the results of treatment with a double reporter control group or PSMA3-deficient HCT-8 with each dose of LPS (B), MDP (C) or TNF-α (D) for 72 hours. After exposure, SEAP and intracellular luciferase levels in the medium were measured simultaneously.
Figure 2 shows the effect of 8-keto-tricotecene on the dual reporter system in human IECs. (AD) Results of treatment of control or PSMA3-deficient HCT-8 with dual reporters for 24 h with each dose of DON (A), 15-acDON (B), Fu-X (C) or NIV (D) indicates. After exposure, SEAP and intracellular luciferase levels in the medium were measured simultaneously.

Accordingly, the present inventors developed an intestinal cell-based bioassay system for measuring the epithelial stress response to 8-keto-tricotecene. In the present invention, EGR-1 was measured as an important biomarker in epithelial exposure to 8-keto-tricotecene. In combination with NF-κB, a representative inflammatory biomarker, an EGR-1-reporter system optimized in human intestinal cells, which responds to unavoidable tricotecene, was constructed and completed the present invention. The present invention can be provided as a biomaterial useful for measuring the initial biological activity of pro-inflammatory tricotecene in food, feed and the environment.

The present invention relates to a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and a cell-based 8-keto-trichothecene detection system comprising a second reporter recombinant vector comprising a p65 promoter gene and a secreted alkaline phosphatase (SEAP) reporter gene. .

Preferably, the EGR-1 binding site gene may be represented by SEQ ID NO: 1 (5'-CGCCCTCGCCCCCGCGCCGGG-3'), but is not limited thereto.

Preferably, the p65 promoter gene may be a human p65 promoter (-999/+92) region, but is not limited thereto.

Preferably, the cells may be intestinal epithelial cells (IECs), and more preferably, proteasome subunit alpha type-3 (PSMA3)-deficient intestinal epithelial cells. may be, but is not limited thereto.

Preferably, the 8-keto-tricotecene is deoxynivalenol (DON), 15-acetyl deoxynivalenol (15-acDON), 3-acetyl deoxynivalenol (3-acetyl deoxynivalenol; 3-acDON), fusarenon-X (Fu-X), nivalenol (NIV), T-2 toxin, aflatoxin B1 and ochratoxin A ( It may be any one or more selected from the group consisting of ochratoxin A), but is not limited thereto.

Preferably, the detection system measures the inflammatory response to 8-keto-tricotecene.

In the present invention, the "reporter gene" refers to a gene whose expression can be easily confirmed visually by exhibiting bioluminescence, fluorescence, or color development during gene expression.

In the present invention, "recombinant vector" means a DNA preparation having a DNA sequence operably linked to suitable regulatory sequences capable of expressing DNA in a suitable host, a plasmid, phage particle, or simply a potential genomic insert. can be Upon transformation into an appropriate host, the recombinant vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.

In addition, the present invention provides: 1) a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and transfecting intestinal epithelial cells with a second reporter recombinant vector comprising a p65 promoter gene and a secreted alkaline phosphatase (SEAP) reporter gene; 2) processing the sample to the transfected intestinal epithelial cells; and 3) measuring LUC activity and SEAP activity in intestinal epithelial cells treated with the sample.

Preferably, the EGR-1 binding site gene may be represented by SEQ ID NO: 1 (5'-CGCCCTCGCCCCCGCGCCGGG-3'), but is not limited thereto.

Preferably, the p65 promoter gene may be a human p65 promoter (-999/+92) region, but is not limited thereto.

Preferably, the intestinal epithelial cell may be a PSMA3-deficient intestinal epithelial cell, but is not limited thereto.

Preferably, in the case of the sample in which the LUC activity is increased and the SEAP activity is decreased compared to the control in which the sample is not treated in step 3), it can be determined that 8-keto-tricotecene is contained.

Preferably, the 8-keto-tricotecene is deoxynivalenol (DON), 15-acetyl deoxynivalenol (15-acDON), 3-acetyl deoxynivalenol (3-acetyl deoxynivalenol; 3-acDON), fusarenon-X (Fu-X), nivalenol (NIV), T-2 toxin, aflatoxin B1 and ochratoxin A ( It may be any one or more selected from the group consisting of ochratoxin A), but is not limited thereto.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

The following experimental examples are provided to provide experimental examples commonly applied to each embodiment according to the present invention.

< Experimental example >

1. Cell Culture Conditions and Reagents

The intestinal epithelial cell line HCT-8 (passage 13) was purchased from ATCC (Rockville, MD, USA). HCT-8 cells were 10% (v/v) heat-inactivated fetal bovine serum (FBS, Welgene), 50 units/ml penicillin and 50 μg/ml streptomycin (Welgene) supplemented with RPMI 1640. medium (Welgene, Daegu, South Korea), 5% CO ₂ was maintained at 37 ℃ in a humid incubator. Cell counting was performed by trypan blue dye exclusion (Sigma-Aldrich chemical company, St. Louis, MO, USA) using a hemocytometer. Except for TNF-α (Peprotech, Rocky Hill, NJ, USA), all other compounds were purchased from Sigma-Aldrich.

2. Plasmid Construction

An empty vector or PSMA3 shRNA (shPSMA3) was constructed using the pMKO.1 GFP vector purchased from Addgene. The inserted PSMA3 shRNA targets the following sequence: 5'-TAA AGC TTT TGA ACT AGA A-3'. As a luciferase reporter construct, a promoter including four EGR-1 binding sites was used. Human p65 promoter (-999/+92) was generated by RT-PCR using human genomic DNA derived from HCT-8 cells. The primers used are as follows. 5'-AAG ACG AAT TCA GAG AAA ATA AAA A-3' and 5'-TGC ACT ACA GAC GAG CCA TT-3'. The resulting 1091 bp construct was inserted into a reporter system with a SEAP gene cassette.

3. luciferase analysis

Cells were washed with cold PBS, lysed with manual lysis buffer (Promega), and then centrifuged at 13,800×g for 10 minutes. The supernatant was collected and stored at -80°C until measurement. The supernatant (10 μl) was briefly mixed with 40 μl firefly luciferase assay substrate solution and 50 μl Renilla After stopping with luciferase stopping solution (Promega), luciferase activity was measured with a model TD-20/20 dual mode luminometer (Turner Designs, Sunnyvale, CA). For standardization, firefly luciferase activity was compared with Renilla split with luciferase.

4. Secretion alkaline Phosphatase (Secreted alkaline) phosphatase ; SEAP ) analysis

Transformed HCT-8 cells were seeded ^{in 24-well plates at a density of 5×10 4} cells/well, and then treated with various concentrations of mycotoxin and other compounds at 37° C. for 24 or 72 hours. After heating the collected culture medium (400 μl) at 65° C. for 5 minutes, 100 μl of the heated supernatant was _{mixed with 100 μl of 2×SEAP assay buffer (2 M diethanolamine, 1 mM MgCl 2} , 20 mM L-homoarginine). was mixed with The mixture was reacted at 37° C. for 10 minutes, and 20 μl of 120 mM p-nitrophenylphosphate dissolved in 1×SEAP assay buffer was added to terminate the reaction. The final mixture was further reacted in the dark at 37°C. Using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA), the absorbance of the reaction mixture was measured at 405 nm.

5. Statistical and reproducibility

GraphPad Prism v. Statistical analysis was performed using 8.01 (La Jolla, CA, USA). For comparative analysis of the two data groups, Student's t- test was performed. For comparative analysis of several groups, data were applied to analysis of variance (ANOVA) using Newman- Keuls method as post hoc ANOVA evaluation. All in vitro evaluations are representative of 2-3 independent experiments. Details of the number of biological replicates and analysis are described in the legend of each figure.

< Example 1> 8- in inflammation control Keto - tricotesen -Dual reporter system that exhibits specific patterns

We found that 8-keto-tricotecene induced EGR-1 in IECs, while repressing p65 promoter activity. We also confirmed that PSMA3 deficiency in IECs enhances EGR-1 levels in response to 8-keto-tricotecene. Based on the above results, the present inventors constructed a cell-based bioassay system representative of the proinflammatory action of 8-keto-tricotecene in IECs. Control and PSMA3-deficient cells were stably transfected with a dual reporter plasmid consisting of a SEAP reporter plasmid containing ^{pEBS1 4 -LUC and human p65 promoter (-999/+92).} The pEBS1 ⁴ -LUC plasmid was constructed to detect EGR-1-mediated transcriptional activity, and by measuring SEAP in the medium, p65 promoter activity was quantified in response to inflammagens (FIG. 1A). In addition, the present inventors analyzed the biological response to cytokines or bacterial endotoxins as positive pro-inflammatory triggers in a double reporter living organism. We calculated an effective amount to enhance reporter production by 50% (ED ₅₀ ) or an inhibitory amount to decrease reporter production by 50% (ID ₅₀ ) in a dual reporter system. In response to bacterial cell wall components comprising LPS or MDP (minimum bioactive peptidoglycan motif), or tumor necrosis factor-α (TNF-α), EGR-1-mediated transcription and Both p65 promoter activity was elevated in a dose-dependent manner ( FIGS. 1B to 1D ). Moreover, PSMA3 deficiency improved the dose response to EGR-1-mediated transcription for traditional pro-inflammatory triggers, particularly MDP, compared to controls. In comparison, PSMA3 deficiency attenuated p65 promoter activation in response to three types of inflammagens, indicating that PSMA3 is a positive regulator of NF-κB signaling activation by endotoxin or TNF-α. . In response to 8-keto-tricotecene, EGR-1-mediated transcription was elevated, but p65 promoter activity was suppressed in a dose-dependent manner ( FIG. 2 ). Moreover, the sensitivity of EGR-1-mediated transcription in response to 8-keto-tricotecene was enhanced by PSMA3 deficiency. However, the 50% inhibitory dose (ID ₅₀ ) for p65 promoter activity was not significantly changed by PSMA3 deficiency in the presence of 8-keto-tricotecene. In conclusion, the dual reporter system in PSMA3-deficient IECs could biomonitor the typical inflammatory regulation by 8-keto-tricotecene and distinguish it from the response of traditional proinflammatory triggers including endotoxins and cytokines.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Enterocyte-based Quantitative Combination of Proinflammatory Sentinels Specific to 8-keto-Trichothecene <130> ADP-2020-0297 <150> KR 10-2020-0063641 <151> 2020-05-27 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EGR-1 binding sites <400> 1 cgccctcgcc cccgcgccgg g 21

Claims (11)

a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and
A cell-based 8-keto-trichothecene detection system comprising a p65 promoter gene and a second reporter recombinant vector comprising a secreted alkaline phosphatase (SEAP) reporter gene. The cell-based 8-keto-tricotecene detection system according to claim 1, wherein the EGR-1 binding site gene is represented by SEQ ID NO: 1. The cell-based 8-keto-tricotecene detection system according to claim 1, wherein the cells are intestinal epithelial cells (IECs). The cell-based 8-keto-tricotecene detection system according to claim 3, wherein the cell is a proteasome subunit alpha type-3 (PSMA3)-deficient intestinal epithelial cell. According to claim 1, wherein the 8-keto-tricotecene is deoxynivalenol (deoxynivalenol; DON), 15-acetyl deoxynivalenol (15-acetyl deoxynivalenol; 15-acDON), 3-acetyl deoxyni valenol (3-acetyl deoxynivalenol; 3-acDON), fusarenon-X (Fu-X), nivalenol (NIV), T-2 toxin, aflatoxin B1 and ochratoxin A cell-based 8-keto-tricotecene detection system, characterized in that at least one selected from the group consisting of A (ochratoxin A). The cell-based 8-keto-tricotecene detection system according to claim 1, wherein the detection system measures an inflammatory response to 8-keto-tricotecene. 1) a first reporter recombinant vector comprising an EGR-1 binding site gene and a luciferase (LUC) reporter gene; and transfecting intestinal epithelial cells with a second reporter recombinant vector comprising a p65 promoter gene and a secreted alkaline phosphatase (SEAP) reporter gene;
2) processing the sample to the transfected intestinal epithelial cells; and
3) A cell-based 8-keto-tricotecene detection method comprising the step of measuring LUC activity and SEAP activity in intestinal epithelial cells treated with the sample. 8. The method of claim 7, wherein the EGR-1 binding site gene is represented by SEQ ID NO: 1. Cell-based 8-keto-tricotecene detection method. The method of claim 7, wherein the intestinal epithelial cells are PSMA3-deficient intestinal epithelial cells. The method according to claim 7, wherein in the case of the sample measured to have increased LUC activity and decreased SEAP activity compared to the control group not treated with the sample in step 3), determining that 8-keto-tricotecene is contained A cell-based 8-keto-tricotecene detection method characterized. The method of claim 7, wherein the 8-keto-tricotecene is deoxynivalenol (DON), 15-acetyl deoxynivalenol (15-acetyl deoxynivalenol; 15-acDON), 3-acetyl deoxyni valenol (3-acetyl deoxynivalenol; 3-acDON), fusarenon-X (Fu-X), nivalenol (NIV), T-2 toxin, aflatoxin B1 and ochratoxin A cell-based 8-keto-tricotecene detection method, characterized in that at least one selected from the group consisting of A (ochratoxin A).

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