Application of beta-sitosterol in preparation of vomitoxin detoxification agent

Abstract

The invention relates to the technical field of preparation of vomitoxin detoxification agents, in particular to application of beta-sitosterol in preparation of a vomitoxin detoxification agent. The invention discloses application of beta-sitosterol in preparation of a vomitoxin detoxification agent. Experimental results show that the beta-sitosterol can relieve the toxicity of the vomitoxin, and the toxicity of the vomitoxin can be possibly relieved by relieving the Class III PI3K / Beclin-1 autophagy pathway of cell autophagy.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of detoxifying agents for vomitoxin, and in particular to application of beta-sitosterol in the preparation of detoxifying agents for vomitoxin. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a type B trichothecene mycotoxin produced by Fusarium graminearum and Fusarium luteum, the pathogens of fusarium head blight. DON is the most common Fusarium toxin contaminating cereal crops such as wheat and corn.

[0003] In the existing technology, detoxification can be carried out to control the DON concentration, which can reduce the economic losses caused by DON pollution. Common DON detoxification methods include physical detoxification, chemical detoxification and biological detoxification. Physical detoxification mainly includes ultrasonic treatment, cleaning, ultraviolet radiation, adsorption, etc. In daily life, farmers often use methods such as cleaning and drying. Chemical detoxification uses certain chemical technologies or substances to achieve the degradation of DON toxins, thereby eliminating the hazards of DON toxins. It mainly includes electrochemical oxidation and alkaline treatment. Biological detoxification mainly includes microbial degradation, enzyme treatment and biotransformation, but at present, because the biosafety, degradation pathways and amplification technology of some degrading bacteria still need further research, biological detoxification is more difficult.

[0004] β-Sitosterol is a tetracyclic triterpenoid compound based on cyclopentaneperhydrophenanthrene. It is a white, amorphous powder at room temperature with a molecular weight of 414.71. It is insoluble in water, slightly soluble in ethanol and acetone, and soluble in organic solvents such as benzene. Plants synthesize β-sitosterol through two metabolic pathways: mevalonic acid and methyl-D-erythritol phosphate. Animals cannot synthesize β-sitosterol. β-sitosterol is the primary phytosterol in human diet, accounting for over 65%. Phytosterols are widely found in various vegetable oils, nuts, fruits, and vegetables, with vegetable oils being the primary source. In addition, some Chinese herbal extracts also contain phytosterols, with β-sitosterol comprising the largest proportion of these phytosterols.

[0005] β-Sitosterol is a safe, green, natural, and effective nutritional supplement. It is non-genotoxic, non-cytotoxic, and widely found in nature. Its properties and physiological functions have led to its widespread application. In medicine, β-sitosterol has a wide range of therapeutic effects. In addition to showing great promise in treating cancer, neurodegenerative diseases, mental illness, and inflammation, it also has potential in treating androgenic alopecia and osteoporosis. In the food industry, β-sitosterol is used as a gelling agent in pigs, chickens, aquatic animals, and foods. Furthermore, adding β-sitosterol / γ-oryzanol to foods as a gelling agent can improve the taste of foods like ham and biscuits. In livestock production, it is primarily added to livestock diets to enhance their growth performance through its physiological functions.

[0006] To investigate whether β-sitosterol, a common plant sterol in the diet, can mitigate the harmful effects of DON toxicity on intestinal health, this study conducted controlled experiments using different concentration gradients of DON and DON-β-sitosterol interactions to verify its ability to mitigate DON toxicity. Furthermore, the mechanism by which β-sitosterol mitigates DON toxicity through cellular autophagy was further explored. Therefore, exploring whether β-sitosterol can mitigate the harmful effects of DON toxicity on human health is of great significance. Summary of the Invention

[0007] The object of the present invention is to provide the use of β-sitosterol in the preparation of a detoxifying agent for vomitoxin.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides the use of beta-sitosterol in preparing a detoxifying agent for vomitoxin.

[0010] Preferably, the β-sitosterol can improve the damage of vomitoxin to intestinal epithelial cells and small intestinal villi, prevent intestinal cell apoptosis, and release inflammatory cytokines.

[0011] Preferably, the β-sitosterol can alleviate the cytotoxicity caused by vomitoxin.

[0012] Preferably, the β-sitosterol reduces the toxicity of vomitoxin via the Class III PI3K / Beclin-1 autophagy pathway.

[0013] The present invention also provides the use of beta-sitosterol in preparing a medicine for preventing or treating vomitoxin poisoning.

[0014] The present invention also provides a detoxification agent for vomitoxin, comprising the β-sitosterol.

[0015] Preferably, the amount of β-sitosterol in the detoxification agent for vomitoxin is 20-100 μg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This study aims to explore whether β-sitosterol, a common dietary substance, can alleviate the threat posed by DON toxicity to human health, as well as the biological function of β-sitosterol in Caco-2 cells. Furthermore, the study explored the molecular mechanism by which β-sitosterol alleviates DON toxicity in Caco-2 cells. The experimental results showed that β-sitosterol promoted the increase of cell activity and the level of intracellular reactive oxygen species, mitochondrial membrane potential, and Ca 2+ Levels of β-sitosterol showed a favorable trend for cell growth. β-Sitosterol significantly inhibited apoptosis and improved oxidative stress in Caco-2 cells, reducing inflammation and significantly inhibiting autophagy. Furthermore, experimental results showed that β-sitosterol significantly downregulated the expression of type III phosphatidylinositol 3-kinase (Class III PI3K), the autophagy positive regulator Beclin-1, and the autophagy substrate p62 protein. Therefore, β-sitosterol can mitigate the toxicity of vomitoxin, potentially alleviating the cytotoxicity of vomitoxin by alleviating the autophagy-related Class III PI3K / Beclin-1 autophagy pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 DON concentration optimization (A, B), β-sitosterol concentration optimization (C), and comparison of β-sitosterol intervention modes (D, E, F). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 2 The effect of β-sitosterol intervention on DON-mediated Caco-2 cell morphology.

[0020] Figure 3 Effects of DON treatment on intracellular ROS levels, mitochondrial membrane potential and Ca 2+ A: Reactive oxygen species detection; B: Mitochondrial membrane potential detection; C: Ca 2+ Concentration detection.

[0021] Figure 4 Effects of β-sitosterol intervention on the oxidative stress and inflammation levels of Caco-2 cells, SOD, T-AOC, CAT, POD, GSH-Px, GSSG / T-GSH, MDA and LDH (A~H), IL-1β, IL-6, IL-10 and TNF-α (I~L).

[0022] Figure 5Effects of β-sitosterol on DON-mediated apoptosis of Caco-2 cells, Calcein / PI live-dead cell fluorescence staining (A) and FITC / PI double staining flow cytometry (B).

[0023] Figure 6 Electron microscopic characterization of the effect of β-sitosterol on DON-induced autophagosome production in Caco-2 cells, with the red arrows indicating autophagosomes (scale bar: 500 nm).

[0024] Figure 7 Cell immunofluorescence images and relative fluorescence quantitative graph G (scale bar: 20 μm) of the effects of β-sitosterol on the expression of autophagy molecules Class III PI3K (A, D), Beclin-1 (B, E) and p62 (C, F) proteins.

[0025] Figure 8 The repair effect of β-sitosterol intervention on DON-induced jejunal epithelial tissue damage in C57BL / 6 mice (scale bar; 100 μm). DETAILED DESCRIPTION

[0026] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Materials:

[0029] Human colon cancer cells (Caco-2), DON (purity ≥ 99%), culture medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), trypsin (containing EDTA), phosphate buffered saline (PBS), cell viability kit (CCK-8), cell reactive oxygen species detection kit (DCFH-DA), Ca 2+ Kit, Annexin V-FITC apoptosis detection kit, mitochondrial membrane potential detection kit (JC-1), Hoechst 33342 staining solution, Calcein-PI cell viability and cytotoxicity kit, superoxide dismutase (SOD) typing test kit, total antioxidant capacity (T-AOC) test kit, catalase (CAT) test kit, total glutathione (GSH-Px) test kit, peroxidase (POD) test kit, total glutathione / oxidized glutathione (GSSG / T-GSH) test kit, cell malondialdehyde test kit (MDA), lactate dehydrogenase test kit (LDH), interleukin-1β (IL-1β) test kit, interleukin-6 (IL-6) test kit, interleukin-10 (IL-10) test kit, tumor necrosis factor-α (TNF-α) test kit, and primers were synthesized by Shanghai Shenggong Biotechnology.

[0030] 2. Cell Viability Assay

[0031] (1) The effect of DON degradation products on cell activity was observed using the CCK-8 method.

[0032] Passageable Caco-2 cells were plated at a density of 4500 cells / well in a 96-well cell culture plate and cultured for 24 hours. A blank group (no cells added), a control group (cells added without treatment), and an experimental group (DON concentrations of 0.1, 0.5, 1, 5, 10, 20, and 50 ppm) were set up, with three replicate wells per group. 100 μL of complete culture medium was added to each well and cultured for 48 hours until the cells reached 70-80% confluence. The culture medium was then removed and serum-free medium containing DON was added. The blank group, without cells, served as a control, while the control group received only DMEM medium and continued to culture for 24 hours. Another group was incubated for 48 hours. Then, 10 μL of CCK-8 reagent was added to each well. After a further 3 hours of incubation, the absorbance of each well at 450 nm was measured using a microplate reader.

[0033] Cell survival rate (RGR) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%. Note: The optimal reaction time of CCK-8 is based on the specific color development time.

[0034] (2) Observation of the effect of β-sitosterol concentration on cell activity according to the CCK-8 method

[0035] The method was the same as (1), and a blank group (no cells added), a control group (cells added without treatment), and an experimental group (β-sitosterol concentrations of 1, 5, 10, 25, 50, 75, and 100 ppm) were set up.

[0036] (3) Observe the effects of different intervention modes on cell activity according to the CCK-8 method

[0037] The method was the same as (1). Different intervention modes were set up and their detoxification effects were tested. The blank group was added with β-sitosterol only, the control group was added with DON only, and the experimental group was divided into three groups: a mixed group of DON and β-sitosterol (DON vs β-sitosterol), a group that added DON first and then β-sitosterol (DON+β-sitosterol), and a group that added β-sitosterol first and then DON (β-sitosterol+DON).

[0038] Results: Effects of different concentrations of β-sitosterol on cell viability

[0039] In this experiment, the CCK-8 assay was used to measure cell viability at various concentrations of β-sitosterol, various concentrations of DON, and different DON intervention modes. The optimal experimental concentration and intervention mode were determined based on the intensity of cell viability. β-sitosterol has anticancer and other physiological activities, and DON is cytotoxic to cells. Therefore, optimizing its concentration is crucial for studying β-sitosterol's DON-mediated cytotoxicity in Caco-2 cells.

[0040] according to Figure 1 In analysis A and B, Caco-2 cells were incubated with different concentrations of DON for 24 and 48 hours, respectively. After 24 hours of DON treatment, cell viability gradually decreased with increasing DON concentration. After 48 hours of DON treatment, cell viability decreased with increasing DON concentration. However, there was no significant difference in cell viability between 1 ppm and 5 ppm DON concentrations. Therefore, the present invention selected 1 ppm as the DON treatment concentration.

[0041] according to Figure 1 C analysis showed that after treating cells with different concentrations of β-sitosterol, when the β-sitosterol concentration was 0-50 ppm, the cell activity gradually increased with the increase of β-sitosterol concentration, while when the β-sitosterol concentration was 50-100 ppm, the cell activity gradually decreased with the increase of β-sitosterol concentration. Therefore, the present invention selected a concentration of 50 ppm (50 μg / ml) of β-sitosterol, which had the highest cell activity.

[0042] according to Figure 1 D, E, and F analyses show that the cell activity of the intervention mode of β-sitosterol followed by DON is higher than that of the intervention mode in which β-sitosterol and DON are added simultaneously and the intervention mode in which DON is added first and then β-sitosterol is added. Therefore, the present invention selects the intervention mode of DON followed by β-sitosterol. In summary, the DON concentration is optimized to be 1ppm according to cell activity, and the β-sitosterol concentration is optimized to be 50μg / mL; the detoxification effects of different intervention modes are compared, and it is found that the prevention mode (β-sitosterol incubation first, DON stimulation later) is better.

[0043] 3. Effects of β-sitosterol intervention on DON-mediated Caco-2 cell morphology

[0044] Passageable Caco-2 cells were plated at 4500 cells / well in a 96-well plate and cultured for 24 hours. A blank control group, a β-sitosterol group, a DON group, and a β-sitosterol + DON group were set up in triplicate. The blank control group was treated with DMEM, the β-sitosterol group was treated with an optimized β-sitosterol concentration, the DON group was treated with an optimized DON concentration, and the β-sitosterol + DON group was treated with an optimized β-sitosterol concentration. After removing β-sitosterol, the optimized DON concentration was added. Finally, the cells were observed under a bright-field microscope.

[0045] Results: In this experiment, the optimized concentration and intervention mode of β-sitosterol first and then DON were set to treat Caco-2 cells. Figure 2 , there was little difference in cell morphology and the number of adherent cells between the blank group and the β-sitosterol group. Compared with the DON group, the blank group showed obvious wrinkling in cell morphology and a significant decrease in cell number in the DON group. Compared with the blank group and the β-sitosterol group, the intervention mode β-sitosterol + DON group showed wrinkling at the cell edges and a decrease in adherent cells, but compared with the DON group, its morphology tended to be more conducive to cell growth and the number of cells was greater. In summary, β-sitosterol intervention improved the DON-induced amoeboid changes in Caco-2 cells, normalized the cell morphology, and increased the number of adherent cells.

[0046] 4. Detection of reactive oxygen species levels, mitochondrial membrane potential and Ca 2+ Release level

[0047] Figure 3 The levels of reactive oxygen species, mitochondrial membrane potential and Ca in Caco-2 cells after β-sitosterol intervention 2+ Changes in concentration. Reactive oxygen detection: The DCFH-DA fluorescent probe was used for reactive oxygen detection. Caco-2 cells were plated in a 96-well cell culture plate at a density of 5000 cells / well and cultured for 24 hours. A blank control group, β-sitosterol, DON group, and β-sitosterol + DON group were set up. Wash with serum-free DMEM medium three times to fully remove excess DCFH-DA probe, and observe the intensity of green fluorescence under an inverted fluorescence microscope. Mitochondrial membrane potential detection: The JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential of Caco-2 cells, and the grouping was the same as above. 100 μL of complete cell culture medium was added to each well, followed by 100 μL of JC-1 staining working solution, mixed thoroughly, and placed in a cell culture incubator for incubation at 37°C for 20 minutes. Hoechst 33342 was added to stain the cell nucleus, washed twice with staining buffer, and then 100 μL of phenol red-free DMEM medium was added. Red and green fluorescence were observed under an inverted fluorescence microscope. Ca 2+Concentration Detection: Caco-2 cell calcium levels in each group were measured using the Fluo-4AM fluorescent calcium probe. Groups were treated as above. Cells were washed three times with PBS to remove residual serum and phenol red. Fluo-4AM staining solution was then added and incubated at room temperature for 30 minutes to allow probe loading. Cells were then washed three times with PBS to remove excess fluorescent probe. Finally, changes in green fluorescence were observed under a fluorescence microscope.

[0048] 5. Effects of β-sitosterol intervention on oxidative stress and inflammation levels in Caco-2 cells

[0049] Test kits for superoxide dismutase (SOD), total antioxidant capacity (T-AOC), catalase (CAT), and total glutathione peroxidase (GSH-Px) were used to measure these indicators. Passage-ready Caco-2 cells were plated in cell culture dishes and cultured for 24 hours. The culture medium was removed and replaced with serum-free DMEM containing DON for another 24 hours. All cells from each dish were trypsinized and collected. The cells were thoroughly lysed using RAPI Lysis Buffer (Strong), centrifuged at 8000 rpm for 10 minutes, and the supernatant was aspirated and stored in aliquots at -80°C until further use. The activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured according to the instructions of each kit.

[0050] Results: SOD, CAT, POD and GSH-Px are important enzyme systems for clearing ROS and maintaining redox homeostasis in cells. In addition, interleukin-6 and interleukin-10 are important inflammatory factors in cells. The above enzyme systems and cytokines play an important role in ensuring the normal physiological function of cells. Figure 4 (A~H) It can be seen that compared with the DON group, the β-sitosterol intervention group increased the activities of intracellular antioxidant enzymes such as SOD, T-AOC, CAT, POD, and GSH-Px, while compared with the DON group, the β-sitosterol intervention group decreased the activities of GSSG / T-GSH, MDA, and LDH. Figure 4 (I, J, K, L) It can be seen that compared with the DON group, the secretion of inflammatory factors such as IL-1β, IL-6, IL-10 and TNF-α in the β-sitosterol intervention group showed a downward trend.

[0051] In summary, β-sitosterol intervention increased the activities of intracellular antioxidant enzymes such as SOD, T-AOC, and CAT, and reduced the GSSG / T-GSH ratio and lipid peroxide (MDA) levels, thereby improving the level of cellular oxidative stress. It also reduced the secretion levels of inflammatory factors such as IL-1β, IL-6, IL-10, and TNF-α, thereby improving the cellular inflammatory state.

[0052] 6. Calcein / PI live-dead cell staining and Annexin V-FITC / PI double staining flow cytometry

[0053] (1) Calcein / PI live-dead cell staining

[0054] Calcein / PI cell viability and cytotoxicity assays were used to assess DON-induced esterase activity and membrane integrity in Caco-2 cells. Passageable Caco-2 cells were seeded into 96-well cell culture plates at 5,000 cells / well and cultured for 48 hours until the cell confluence reached 70% to 80%. A control group (no treatment) and experimental groups (β-sitosterol, β-sitosterol + DON, and DON) were set up. The cell culture medium was removed, and DON was added for 6 hours. Then, 100 μL of Calcein AM / PI working solution was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. After incubation, the staining effects were directly observed under a fluorescence microscope.

[0055] (2) Annexin V-FITC / PI double staining

[0056] Cell apoptosis was assessed using Annexin V-FITC and PI double staining. Groups were divided as above, and then the culture medium was removed and replaced with serum-free DMEM containing DON for another 24 hours. The cell culture medium was removed, and the appropriate volume of Annexin V-FITC conjugate, Annexin V-FITC, and propidium iodide staining solution was added to each group. The cells were incubated in the dark for 15 minutes, shielded from light using aluminum foil.

[0057] Results: In this experiment, Calcein / PI was used to stain live and dead cells, and Annexin V-FITC / PI double staining flow cytometry was used to detect cell apoptosis. Figure 5 A, There was no significant difference in the intensity of CalceinAM green fluorescence and PI red fluorescence between the blank group and the β-sitosterol group. The green fluorescence of the DON group was weakened and the red fluorescence intensity was significantly enhanced compared with the other groups. The green fluorescence of the β-sitosterol + DON intervention group was enhanced and the red fluorescence intensity was significantly weakened compared with the DON group, indicating that the number of dead cells was reduced. Figure 5B, Q1 is cell fragments without cell membrane, Q2 is late apoptotic cells or necrotic cells, Q3 is early apoptotic cells, and Q4 is normal living cells. Comparing Q2 in each group, DON had the most late apoptotic cells or necrotic cells, accounting for 33.8% of the total cell number. Compared with the blank group, the Q2 of the β-sitosterol group was 7.31% and 6.12%, respectively, with no significant difference. Compared with the DON group, the Q2 of the β-sitosterol + DON intervention group was 21.4% and 31.4%, respectively. It can be seen that the number of dead cells was significantly reduced after β-sitosterol intervention. In summary, Calcein / PI live and dead cell staining and AnnexinV-FITC / PI double staining flow cytometry showed that β-sitosterol reduced the degree of DON-mediated cell apoptosis.

[0058] 7. Biological electron microscopy

[0059] The Caco-2 cells to be passaged were transferred into a 6-well plate at 45,000 cells / well and cultured for 24 hours. A control group (no treatment) and an experimental group (β-sitosterol group, β-sitosterol + DON, DON) were set up, and then the cell culture medium was removed. β-sitosterol and DON were added to treat the cells in sequence. After the cells were treated, they were first fixed with glutaraldehyde. In order to maintain the authenticity of the sample, PBS was rinsed 3 times, and a certain amount of electron microscopy fixative was added. The cells were fixed at 4°C for 3.5 hours. Then, the cells were embedded, and the embedding medium was able to evenly support the sample. Ultrathin sectioning was performed, and the embedded cell clusters were cut into thin slices of 50-60 nm. Finally, the samples were stained with heavy metals for about 30 minutes, kept in the greenhouse overnight, and then observed under a lens electron microscope.

[0060] Results: According to Figure 6 Analysis and transmission electron microscopy showed that in the blank group, only some organelles were ruptured and very few vesicle-like structures appeared in the cells. The number of vesicle-like structures in the β-sitosterol group was not much different from that in the blank group. After treatment with the DON group, a large number of vesicle-like structures were observed in the organelles. The vesicles were irregular in shape and contained substances inside. It is speculated that the inside of them were other cellular components or broken organelles, and it was preliminarily judged that the above vesicles were autophagic vesicles. Compared with the DON group, the number of vesicle-like structures in the β-sitosterol + DON intervention model group was significantly reduced. It is speculated that β-sitosterol greatly reduced the production of vesicles and the damage of other components or organelles in the cells. In summary, biological electron microscopy showed that β-sitosterol intervention significantly reduced the production of intracellular autophagosomes induced by DON.

[0061] 8. Cell Immunofluorescence and qRT-PCR

[0062] (1) Cell immunofluorescence

[0063] Immunofluorescence was used to analyze the expression levels of class III phosphatidylinositol 3-kinase (PI3K), the autophagy positive regulator Beclin-1, and the autophagy substrate p62. Sample preparation for immunofluorescence was similar to that for biological electron microscopy, including fixation, embedding, ultrathin sectioning, and staining. Paraffin tissue sections were rinsed three times with PBS and then fixed. Antigen retrieval was then performed using antigen retrieval buffer, followed by incubation with a specific fluorescent primary antibody and a labeled fluorescent secondary antibody. Finally, cell nuclei were restained with DAPI at room temperature, and fluorescence images were observed using an anti-quenching mounting medium.

[0064] (2) qRT-PCR

[0065] ① Total RNA extraction from Caco-2 cells

[0066] The expression levels of messenger RNA (mRNA) of autophagy-related genes in Caco-2 cells were determined by reverse transcription polymerase chain reaction (qRT-PCR). Logarithmic-phase cells were seeded at 500,000 cells / dish in 6-cm culture dishes and incubated for 24 hours. The culture medium was removed and the cells were rinsed twice with PBS. A control group (no treatment) and experimental groups (β-sitosterol group, β-sitosterol + DON group, and DON group) were set up.

[0067] Discard the cell culture medium and DON toxin cell culture medium, rinse twice with PBS, lyse the cells with Trizol, mix thoroughly by pipetting, and transfer to a 1.5 mL RNase-free EP tube. Precool the centrifuge to 4°C. Add 400 μL of chloroform (1 / 5 the volume of Trizol). Centrifuge, slowly aspirate the supernatant, transfer to another 1.5 mL RNase-free EP tube, add an equal volume of isopropanol, mix thoroughly by inverting, and let stand at room temperature for 15 minutes. A white precipitate will be visible upon centrifugation. Wash with 75% ethanol, then open the tube and air dry. Dissolve the precipitate in DEPC water according to its size for each sample.

[0068] ②Reverse transcription reaction (TB Green qPCR method)

[0069] After dissolution, measure the RNA purity and concentration using an ultra-micro UV spectrophotometer. Amplify the RNA into cDNA using a PCR instrument to reduce RNA degradation. Prepare a 30 μL reaction system and calculate the amount of cDNA required by the formula (system size × 1000) / sample concentration = required amount of cDNA. Then add 5 × Prime Script TMPrepare 6 μL of RT MasterMix and finally fill the reaction volume to 30 μL with RNase-free ddH2O. PCR primer sequences for each gene are shown in Table 2. Also prepare 10 μL of TB Green premix ExTaq™ II solution, 1 μL of upstream sequence, 1 μL of downstream sequence, 7 μL of ddH2O, and 1 μL of cDNA (for a control, add 1 μL of DEPC water). After preparation, perform RT-PCR using GADPH as an internal control and observe the results.

[0070] Table 1 Gene primer sequences

[0071]

[0072] As shown in SEQ ID NOs: 1 to 8.

[0073] 9. Pathological changes of jejunum in C57BL / 6 mice

[0074] Figure 8 The results of jejunal tissue sections from C57BL / 6 mice are shown. In the control group, intestinal epithelial cells were normal, with tightly packed villi. Compared with the control group, the DON group showed more pronounced intestinal villi fragmentation, with the striated border of the villi disappearing and increased inter-glandular spacing. Compared with the control group, the β-sitosterol group showed improved intestinal epithelial cell status, as evidenced by more intact jejunal mucosal tissue and more tightly packed villi. These results suggest that β-sitosterol can ameliorate DON-induced damage to intestinal epithelial cells and villi, preventing cell apoptosis and the release of inflammatory cytokines.

[0075] Results: The cell immunofluorescence method was used to detect the in situ expression of type III phosphatidylinositol 3-kinase (Class III PI3K), autophagy positive regulatory protein and autophagy substrate p62 protein in cells, and their fluorescence levels are shown in the figure. Figure 7 (A, B, C) Analysis showed that the fluorescence intensity of Class Ⅲ PI3K, Beclin-1, p62 and DAPI was not significantly different between the blank group and the β-sitosterol group. The DON group had the highest fluorescence intensity compared with the other groups. The fluorescence intensity of the β-sitosterol + DON intervention group was significantly lower than that of the DON group. Figure 7 (D, E, F) Analysis showed that when treated with DON alone, the expression levels of Class III PI3K, Beclin-1 and p62 were significantly higher than those in the blank group. After β-sitosterol + DON intervention, the expression levels of Class III PI3K, Beclin-1 and p62 were significantly decreased compared with those in the DON group.

[0076] according to Figure 7G analysis showed no significant differences in the relative fluorescence intensity of Class III PI3K, Beclin-1, and p62 between the blank group and the β-sitosterol group. The DON group had the highest relative fluorescence intensity compared to the other groups. The β-sitosterol + DON intervention group showed a decrease in relative fluorescence intensity compared to the DON group.

[0077] In summary, the results of cell immunofluorescence and relative quantitative fluorescence showed that β-sitosterol intervention significantly reduced the expression levels of type III phosphatidylinositol 3-kinase (Class III PI3K), autophagy positive regulatory protein Beclin-1 and autophagy substrate p62 protein.

[0078] In the present invention, experimental data show that β-sitosterol can reduce the ROS level of Caco-2 cells treated with DON, improve the oxidative stress level, and reduce the secretion level of inflammatory factors.

[0079] In the present invention, β-sitosterol treatment significantly reduced Class III PI3K, Beclin-1 gene, and p62 protein expression levels as analyzed by cell immunofluorescence and RT-PCR, consistent with the above results. Although the Class III PI3K / Beclin-1 signaling pathway is involved in regulating autophagy, the molecular mechanisms of autophagy are complex, and the mechanism by which β-sitosterol alleviates DON toxins requires further exploration.

[0080] In summary, β-sitosterol intervention can reduce DON toxicity through the Class III PI3K / Beclin-1 autophagy pathway, indicating that β-sitosterol may be an important dietary component for preventing DON toxicity from harming the human body.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of β-sitosterol in the preparation of detoxification agent for vomitoxin.

2. The use according to claim 1, characterized in that The beta-sitosterol can improve the damage of vomitoxin to intestinal epithelial cells and small intestinal villi, and prevent cell apoptosis and the release of inflammatory cytokines.

3. The use according to claim 1, characterized in that The β-sitosterol can alleviate the cytotoxicity caused by vomitoxin.

4. The use according to claim 3, characterized in that The β-sitosterol reduces the toxicity of vomitoxin through the Class III PI3K / Beclin-1 autophagy pathway.

5. Use of β-sitosterol in the preparation of drugs for preventing or treating vomitoxin poisoning.

6. A detoxification agent for vomitoxin, characterized in that: Containing the β-sitosterol according to claim 5.

7. The detoxification agent for vomitoxin according to claim 6, characterized in that: The dosage of the beta-sitosterol in the detoxification agent for vomitoxin is 20 to 100 μg / mL.

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