Application of fraxinin in the preparation of antidotes for preventing and treating zearalenone poisoning

Abstract

This invention discloses the application of fraxin in the preparation of an antidote for zearalenone poisoning. Using zebrafish as the experimental subject, this invention found that fraxin has a good detoxification effect on zearalenone. Experiments showed that fraxin can, on the one hand, prevent zearalenone-induced embryonic death in zebrafish, increase the hatching rate of embryos, enhance embryonic activity, and inhibit zearalenone-induced apoptosis, restore cell cycle arrest, and prevent senescence of damaged cells. On the other hand, experiments showed that fraxin can completely reverse the negative effects caused by zearalenone, alleviating the toxicity of zearalenone while promoting the growth of zebrafish larvae.

Description

Technical Field

[0001] This invention relates to the field of mycotoxin control technology, specifically to the application of fraxin in the preparation of an antidote for zearalenone poisoning. Background Technology

[0002] Zearalenone (ZEA) is a fungal toxin commonly detected in corn and other grains. It poses a serious threat to human health and animal husbandry. Also known as F-2 toxin, ZEA is a secondary metabolite produced by Fusarium fungi (such as Fusarium graminearum, Fusarium wilt, Fusarium graminearum, and Fusarium hemifibrillariae), specifically an estrogen-like fungal toxin. It can produce immunotoxicity, cytotoxicity, and genotoxicity, damaging the liver and kidneys, and even leading to death. The direct and indirect losses caused by fungal toxin contamination each year are enormous. Therefore, comprehensive testing of toxin content in grain crops and strict control of ZEA residues to ensure levels remain below the limits are of great significance for grain security and human health.

[0003] Currently known methods for controlling zearalenone include physical, chemical, and biological methods. While these methods can mitigate the effects of zearalenone to varying degrees, they still suffer from incomplete effectiveness and low efficiency. Known antidotes for zearalenone poisoning can reduce its toxicity, but their effectiveness is limited and cannot completely eliminate the risk. Therefore, finding more effective preventative methods is urgently needed. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide the application of fraxin in the preparation of an antidote for zearalenone poisoning, thereby solving the problem of poor efficacy of existing antidotes for zearalenone poisoning.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] Application of fraxin in the preparation of antidotes for zearalenone poisoning.

[0007] An antidote for zearalenone poisoning includes the aforementioned fraxin.

[0008] Furthermore, the concentration of fraxin in the aforementioned antidote for zearalenone poisoning is 20-40 μmol / L.

[0009] The present invention has the following beneficial effects:

[0010] (1) This invention demonstrates through experiments that fraxetin has a good detoxification effect on zearalenone poisoning. On the one hand, fraxetin prevents zebrafish embryo death, increases the hatching rate of embryos, and enhances embryo activity. On the other hand, it also inhibits apoptosis caused by zearalenone, restores cell cycle arrest, and prevents senescence of damaged cells. Moreover, on the other hand, potency analysis based on zebrafish shows that the alleviating potency of fraxetin is 4.57 μmol / L, and its alleviating effect can completely reverse the negative effects of zearalenone at 40 μmol / L (approximately 8.32 mg / kg).

[0011] (2) The present invention also demonstrated through experiments that fraxin can promote the growth of zebrafish while alleviating the toxic effects of zearalenone. Attached Figure Description

[0012] Figure 1 Figure showing the morphological changes induced in zebrafish embryos by fraxinus and zearalenone.

[0013] Figure 2 The diagram shows the dose-response relationship between different concentrations of fraxetin and the alleviation of zearalenone in zearalenone. In this diagram, A represents the deformity rate of fraxetin at different intervention concentrations at 72 hpf; B represents the mortality rate of fraxetin at different intervention concentrations at 72 hpf; C represents the hatching rate of fraxetin at different intervention concentrations at 72 hpf; and D represents the median effective concentration (EC50) of fraxetin, assessed using a toxicity score based on mortality, hatching rate, and deformity rate. 50 The fitted curve;

[0014] Figure 3 Figure showing the effect of fraxin on the body size of zebrafish;

[0015] Figure 4 The graph shows the effect of different concentrations of fraxetin on the growth recovery of zebrafish. In the graph, A represents the body length at 72 hpf and 120 hpf under different intervention concentrations of fraxetin, B represents the heart area at 72 hpf and 120 hpf under different intervention concentrations of fraxetin, C represents the yolk sac area at 72 hpf and 120 hpf under different intervention concentrations of fraxetin, D represents the head height at 72 hpf and 120 hpf under different intervention concentrations of fraxetin, E represents the eye area at 72 hpf and 120 hpf under different intervention concentrations of fraxetin, and F represents the swimming sac area at 72 hpf and 120 hpf under different intervention concentrations of fraxetin.

[0016] Figure 5The results of the analysis of apoptosis induced by zearalenone by fraxin are shown. Among them, A is the number of apoptotic cells in zebrafish indicated by AO staining, B is a statistical graph of the number of apoptotic cells in the head stained by AO, C is a statistical graph of the number of apoptotic cells in the trunk stained by AO, and D is a statistical graph of the number of apoptotic cells in the tail stained by AO.

[0017] Figure 6 The results of the analysis of cell cycle arrest induced by fraxinus reversing zearalenone are shown in Figure A, where flow cytometry cell cycle analysis (24 hpf) using PI staining is shown, and Figure B shows the quantitative results of cell cycle in each group.

[0018] Figure 7 The results of the experiment show that fraxin reverses cellular senescence induced by zearalenone. In the figure, A is a representative image and quantification of cellular senescence at 24 hpf, and B is a representative image and quantification of cellular senescence at 72 hpf.

[0019] Figure 8 The results of experiments showing the reversal of transcriptional profile changes induced by zearalenone by fraxin. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0021] Example 1:

[0022] 1. Experimental equipment

[0023] Stereo microscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan); commercially available 6-well plates; vortex mixing apparatus; ultra-low temperature freezer; constant temperature incubator; zebrafish farming system; thermometer; plastic droppers; general laboratory glassware and equipment.

[0024] 2. Experimental Methods

[0025] (1) Preparation of zebrafish standard culture medium

[0026] Weigh out 1.176 g of calcium chloride, 493 mg of magnesium sulfate, 252 mg of sodium bicarbonate and 22 mg of potassium chloride respectively, dissolve them in water, and dilute to 100 mL with water to prepare calcium chloride stock solution, magnesium sulfate stock solution, sodium bicarbonate stock solution and potassium chloride stock solution.

[0027] Take 25 mL of each of the above stock solutions, mix them together, and then dilute with water to 1000 mL to obtain the standard culture solution.

[0028] (2) Preparation of fraxin culture medium

[0029] Weigh 20 mg of fraxetin, dissolve it in 600.5 μL of dimethyl sulfoxide, and dilute it with standard culture medium to a fraxetin concentration of 20 μmol / L or 40 μmol / L to obtain the solution.

[0030] (3) Wild-type AB strain zebrafish embryos with normal development and 6 hours post-fertilization (6hpf) were randomly selected and placed in 6-well plates, with 20 embryos per well. The following experimental groups were set up: normal control group, zearalenone exposure group and fraxinus intervention group.

[0031] In the zearalenone exposure group, 4 mL of pre-prepared zearalenone exposure solution with a concentration of 2000 μg / L was added to each well.

[0032] In the normal control group, 4 mL of standard culture medium containing 0.05% dimethyl sulfoxide was added to each well;

[0033] In the fraxetin intervention group, 4 mL of a mixed solution of zearalenone exposure solution and fraxetin culture medium was added to each well. The concentration of zearalenone in the mixed solution was 2000 μg / L, and the concentration of fraxetin was 1.25, 2.5, 5, 10, 20 and 40 μmol / L.

[0034] (4) Expose the embryos to 24 hpf and observe the embryo toxicity phenotype under a stereomicroscope.

[0035] (5) Expose the embryos to 72 hpf and observe and record the mortality rate, malformation rate, hatching rate and embryo toxicity phenotype under a stereomicroscope.

[0036] (6) Toxicity score

[0037] The deformities and hatching status of 72hpf larvae were assessed according to the methods described in Table 1. The assessment included the following categories: 1) head abnormalities such as microcephaly, 2) eye abnormalities such as microphthalmia, 3) spinal and tail abnormalities such as spinal curvature or tail twisting, 4) heart and yolk sac abnormalities such as edema, 5) positional abnormalities such as lateral recumbency or inactivity, 6) organ abnormalities such as jaundice, and 7) hatching status. The larval status was assessed according to established criteria, ranging from none (0 points) to severe (6 points), and the overall scores for all categories in each condition were summed to generate a "toxicity score." A higher toxicity score indicates a more pronounced toxic effect.

[0038] Table 1. Detailed Scoring Rules for Zearalenone Toxicity

[0039]

[0040] (7) Conduct valence assessment

[0041] Lethality, hatchability, and malformation data were quantified and scored. At least 20 larvae were analyzed for each treatment group and concentration in the experiment. This invention uses a 4-parameter Hill model to fit a standard sigmoid curve, defining the response based on four parameters {T, B, EC50, Hill slope}. The upper (“Top”) and lower (“Bottom”) asymptotes of each measured response are represented by parameters T and B, respectively. The “Hill slope” determines the slope of the curve between B and T (the change in response relative to concentration), with larger numbers indicating a steeper curve. GraphPad Prism version 9.5.0 (GraphPad Software, Inc., CA, USA) was used for parameter fitting. The alleviating potency of the compound was defined as the calculated half-maximal effective concentration (EC50). 50 value.

[0042] 3. Experimental Results

[0043] Experimental results are as follows Figure 1 and Figure 2 As shown.

[0044] from Figure 1 It is known that the antidote fraxin exhibits a dose-dependent toxicity to zearalenone, and no other significant negative effects were observed in the concentration range of 1.25-40 μmol / L.

[0045] from Figure 2 It can be seen that at concentrations of 20 μmol / L and 40 μmol / L, fraxetin completely eliminates the abnormality rate ( Figure 2 A) Mortality rate ( Figure 2 B) and incubation delay ( Figure 2 C). Based on mortality rate, hatching rate, and deformity rate, the half-maximal effective concentration (EC50) of fraxetin was assessed using a toxicity score. 50 The fitted curve of fraxetin EC 50 The value was 4.57 μmol / L. Figure 2 D), which means that at this concentration, fraxetine can reduce the toxic effects by half.

[0046] In conclusion, fraxin can effectively alleviate the negative effects of zearalenone.

[0047] Example 2:

[0048] 1. Experimental equipment

[0049] Stereo microscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan); commercially available 6-well plates; vortex mixing apparatus; ultra-low temperature freezer; constant temperature incubator; zebrafish farming system; thermometer; plastic droppers; general laboratory glassware and equipment.

[0050] 2. Experimental Methods

[0051] (1) Wild-type AB strain zebrafish embryos with normal development and 6 hours post-fertilization (6hpf) were randomly selected and placed in 6-well plates, with 20 embryos per well. The following experimental groups were set up: normal control group, zearalenone exposure group and fraxinus intervention group.

[0052] in:

[0053] In the zearalenone exposure group, 4 mL of pre-prepared zearalenone exposure solution was added to each well, with a zearalenone concentration of 1500 μg / L;

[0054] In the normal control group, 4 mL of standard culture medium containing 0.05% dimethyl sulfoxide was added to each well;

[0055] In the fraxetin intervention group, 4 mL of a mixed solution of zearalenone exposure medium and fraxetin culture medium was added to each well. The concentration of zearalenone in the mixed solution was 1500 μg / L, and the concentration of fraxetin was 20 μmol / L (ZEA+Fra20) and 40 μmol / L (ZEA+Fra40).

[0056] (2) Expose the embryos to 72 hpf or 120 hpf and observe and record the zebrafish toxic phenotype under a stereomicroscope.

[0057] (3) Use ImageJ software to measure the whole body morphological parameters of zebrafish.

[0058] 3. Experimental Results

[0059] Experimental results are as follows Figure 3 and Figure 4 As shown.

[0060] from Figure 3 It is known that at 72 hpf or 120 hpf, the antidote fraxinus can restore the body size of zebrafish at a concentration range of 20 or 40 μmol / L.

[0061] from Figure 4 It can be seen that at 72 hpf or 120 hpf, the antidote fraxinus, at concentrations of 20 or 40 μmol / L, not only restored the body length of zebrafish but also promoted its growth. Figure 4A) In addition, fraxetin completely restored the heart area, yolk sac area, head height, eye area, and swimming sac area.

[0062] In summary, 20 μmol / L or 40 μmol / L fraxin completely alleviated the growth inhibition caused by zearalenone. In addition, fraxin also produced a growth-promoting effect under the growth-promoting effect.

[0063] Example 3:

[0064] 1. Experimental equipment

[0065] Inverted fluorescence microscope (Leica DMI4000B, Leica Biosystems, Heidelberg, Germany); stereo microscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan); commercially available 6-well plates; vortex mixing apparatus; ultra-low temperature freezer; constant temperature incubator; zebrafish culture system; thermometer; plastic droppers; general laboratory glassware and equipment.

[0066] 2. Experimental Methods

[0067] (1) Wild-type AB strain zebrafish embryos with normal development and 6 hours post-fertilization (6hpf) were randomly selected and placed in 6-well plates, with 20 embryos per well. The following experimental groups were set up: normal control group, zearalenone exposure group and fraxinus intervention group.

[0068] in:

[0069] In the zearalenone exposure group, 4 mL of pre-prepared zearalenone exposure solution was added to each well, with a zearalenone concentration of 1500 μg / L;

[0070] In the normal control group, 4 mL of standard culture medium containing 0.05% dimethyl sulfoxide was added to each well;

[0071] In the fraxetin intervention group, 4 mL of a mixed solution of zearalenone exposure medium and fraxetin culture medium was added to each well. The concentration of zearalenone in the mixed solution was 1500 μg / L, and the concentration of fraxetin was 20 μmol / L (ZEA+Fra20) and 40 μmol / L (ZEA+Fra40).

[0072] (2) Expose the embryos to 24 hpf or 72 hpf and detect various indicators, including apoptosis, cell cycle and cell senescence.

[0073] (3) Apoptosis analysis: Acridine orange (AO) was used to assess apoptosis.

[0074] Live embryos were incubated in standard culture medium containing 2 μg / mL AO (Sigma, St. Louis, MO, USA) at 28°C for 30 minutes. The embryos were washed five times with the standard culture medium to remove excess dye. Fluorescence imaging was performed using a Leica DMI4000B fluorescence microscope (Leica Biosystems, Heidelberg, Germany). Apoptosis counts were analyzed using Aivia 11.0.1 software (Leica Microsystems, Bellevue, WA) and ImageJ software (ImageJ, Maryland, USA).

[0075] (4) Cell cycle analysis

[0076] Preparation of single-cell suspension: Approximately 40 collected embryos were transferred to centrifuge tubes containing DMEM / F12 medium and 10% fetal bovine serum, and single-cell suspensions were obtained by grinding.

[0077] The single-cell suspension was washed with PBS to remove residual culture medium and serum components.

[0078] The washed single-cell suspension was fixed in 70% ethanol at 4°C for 1 hour, and then transferred to -20°C for overnight fixation. This step is used to maintain cell morphology and structure and prepare the cells for staining and analysis.

[0079] The next day, the fixed samples were treated with PI / RNase staining solution (BD Biosciences, San Jose, CA, USA).

[0080] Samples were run on a BD Verse flow cytometer (BD Biosciences, San Jose, CA, USA).

[0081] The collected data were analyzed using ModFitLT software (Verity, Topsham, ME).

[0082] (5) Cell senescence analysis

[0083] Senescence-associated β-galactosidase (SA-β-gal) was detected using a senescence β-galactosidase staining kit (Beyotime, Shanghai, China). Embryos at 24 hpf and 72 hpf were fixed overnight in PBS with 4% paraformaldehyde at 4°C. The embryos were then washed three times with PBS at pH 7.4 for 20 minutes each time, followed by a wash with PBS at pH 6.0 at 4°C for 1 hour. Staining was then performed overnight at 37°C. To quantitatively assess the level of SA-β-Gal (senescence-associated β-galactosidase) in vivo, all larvae were photographed under standardized conditions using a dissecting microscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan). High-resolution digital images were generated to facilitate the selection of stained areas showing visually positive SA-β-Gal staining. Quantitative analysis was performed using Adobe Photoshop software (Adobe Systems, San Jose, CA). Using this software, the percentage of stained pixels was determined by calculating the ratio between the number of stained pixels in each selected area and the total number of pixels. This method can more objectively assess the degree of SA-β-Gal staining in samples, thus enabling precise quantitative comparisons between different experimental treatments.

[0084] 3. Experimental Results

[0085] The experimental results are shown in Figure 5 , Figure 6 , Figure 7 and Figure 8 .

[0086] from Figure 5 It can be seen that fraxin at a concentration of 40 μmol / L can completely restore the apoptosis phenomenon caused by zearalenone.

[0087] from Figure 6 It can be seen that fraxin can completely restore the cell cycle arrest caused by zearalenone at a concentration of 40 μmol / L.

[0088] from Figure 7 It can be seen that fraxin, at a concentration of 40 μmol / L, can completely reverse the cellular senescence caused by zearalenone.

[0089] from Figure 8 It can be seen that fraxinus can restore the changes in the transcriptional pattern of zebrafish induced by zearalenone at a concentration of 20-40 μmol / L, with most genes reaching the level of the normal control group.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of fraxin in the preparation of an antidote for zearalenone poisoning.

2. The application of fraxin according to claim 1 in the preparation of an antidote for zearalenone poisoning, characterized in that, The concentration of fraxetin is 20-40 μmol / L.

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