Acute gastrointestinal toxicity evaluation method based on Caco-2 cell model

Abstract

The invention provides a method for evaluating acute gastrointestinal toxicity of a test substance based on a Caco-2 cell model, which is characterized by comprising the following steps: acquiring Caco-2 monolayer cells; enabling the Caco-2 monolayer cells to be in contact with a test substance; detecting the amount of fluorescein penetrating through the Caco-2 monolayer cells before and after contact; and / or detecting the amount of Evans blue penetrating through the Caco-2 monolayer cells before and after contact; and / or detecting the activity change of lactic dehydrogenase in the Caco-2 monolayer cell external culture medium before and after contact; and evaluating the acute gastrointestinal toxicity of the test substance according to the detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for evaluating acute gastrointestinal toxicity based on a Caco-2 cell model. BACKGROUND

[0002] Currently, there is no unified in vitro evaluation standard and model for acute gastrointestinal toxicity of chemicals and proteins. Traditional animal experiments are long, costly, and controversial in ethics, and it is difficult to achieve high-throughput screening. Caco-2 cells can differentiate into a cell layer with intestinal epithelial characteristics under specific culture conditions and have been widely used in drug absorption and transport research, but their application in acute toxicity evaluation of protein substances has not been systematically reported.

[0003] Therefore, it is of important scientific and practical value to establish a standardized, repeatable, and efficient Caco-2 cell model for evaluating acute gastrointestinal toxicity. SUMMARY

[0004] The purpose of the present application is to provide a method for evaluating acute gastrointestinal toxicity based on a Caco-2 cell monolayer model, which comprehensively evaluates the intestinal toxicity of the test substance by detecting transmembrane electrical resistance (TEER), fluorescence yellow / evans blue permeability, and lactate dehydrogenase (LDH) activity.

[0005] To solve the above problems, the present application provides the following solutions.

[0006] 1. A method for evaluating the acute gastrointestinal toxicity of a test substance based on a Caco-2 cell model, characterized in that it comprises the following steps: obtaining Caco-2 monolayer cells; contacting the Caco-2 monolayer cells with the test substance; detecting the amount of fluorescence yellow that permeates the Caco-2 monolayer cells before and after contact; and / or detecting the amount of evans blue that permeates the Caco-2 monolayer cells before and after contact; and / or detecting the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact; evaluating the acute gastrointestinal toxicity of the test substance according to the detection results.

[0007] 2. The method of item 1, wherein, detecting the amount of fluorescence yellow that permeates the Caco-2 monolayer cells before and after contact and detecting the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact; or detecting the amount of evans blue that permeates the Caco-2 monolayer cells before and after contact and detecting the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact.

[0008] 3. The method according to item 1, wherein, The evaluation of acute gastrointestinal toxicity of test substances based on test results includes the following: Calculate the concentration of fluorescein 染毒后 Evans Blue Concentration 染毒后 Changes in lactate dehydrogenase activity (OD) 染毒后 / OD 染毒前 ; Assessing acute gastrointestinal toxicity: Acute gastrointestinal toxicity is indicated by the presence of one or two of the following: OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of fluorescein in the exposed group 染毒后 Higher than the negative control group; or, OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of Evans blue in the treated group 染毒后 Higher than the negative control group; or, OD 染毒后 / OD 染毒前 And / or the concentration of fluorescent yellow in the poisoned group 染毒后 All were higher than the negative control group; or, OD 染毒后 / OD 染毒前 And / or the concentration of Evans blue in the infected group 染毒后 All were higher than the negative control group.

[0009] The negative control group is the result when the concentration of the test substance is 0.

[0010] 4. The method according to item 1, wherein the contact time between the Caco-2 monolayer cells and the test substance is less than 10 hours; preferably 1-6 hours.

[0011] 5. The method according to item 1, wherein the Caco-2 cells are cultured for at least 21 days before being brought into contact with the test substance; preferably 21-28 days.

[0012] 6. The method according to item 1, wherein the test substance is selected from protein substances, chemicals, drugs or food additives.

[0013] 7. The method according to item 6, wherein the protein is selected from one or both of pollen protein and ricin.

[0014] 8. The method according to item 7, wherein the concentration of the proteinaceous substance is 0.01-50 μg / ml.

[0015] 9. The method according to item 8, wherein the concentration of the proteinaceous substance is 0.1-50 μg / ml when the proteinaceous substance is trypsin inhibitor; and the concentration of the proteinaceous substance is 0.01-20 μg / ml when the proteinaceous substance is ricin.

[0016] 10. The method according to any one of items 1 to 9, wherein the chemical is selected from one or both of 2,3,4,5-tetrabromo-benzene dicarboxylic acid bis(2-ethylhexyl) ester (TBPH) and cadmium chloride (CdCl2).

[0017] 11. The method according to item 10, wherein the concentration of the chemical is 1-160000 nmol / L.

[0018] 12. The method according to item 10, wherein the concentration of the chemical is 1-5000 nmol / L, preferably 1-2000 nmol / L, when the chemical is TBPH; and the concentration of the chemical is 0.01 μM-160 μM, preferably 10 μM-160 μM, when the chemical is cadmium chloride.

[0019] 13. The method according to item 3, wherein the method further comprises a positive control step, and the positive control is 1% Triton-X 100. 14. The method according to item 1, wherein the cells are washed with PBS or HBSS buffer before and after the Caco-2 monolayer cells are contacted with the test substance.

[0020] 15. Use of the acute gastrointestinal toxicity evaluation method according to any one of items 1 to 14 in evaluating gastrointestinal toxicity of a drug, food additive or chemical.

[0021] Compared with the prior art, the application has the following beneficial effects: The application first systematically uses the Caco-2 cell model for acute gastrointestinal toxicity evaluation of proteinaceous substances. The results are more comprehensive and reliable by using multi-index joint evaluation. The method is simple, reproducible and low in cost, and is suitable for high-throughput screening. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A flow chart of the test is shown; Figure 2 ​​This diagram illustrates a Transwell-cultured Caco-2 monolayer model. Figure 3 This study demonstrates the effect of trichosanthes pollen protein exposure on the transmembrane resistance of Caco-2 monolayer cells. (TEER) 染毒后 / TEER 染毒前 ); Figure 4 This study demonstrates the effect of pollen protein exposure on the integrity of Caco-2 monolayer cells. (Concentration of fluorescein in the Transwell outer chamber); Figure 5 This study demonstrates the effect of pollen protein exposure on the integrity of Caco-2 monolayer cells. (Concentration of Evans blue in the Transwell outer chamber); Figure 6 This study demonstrates the effect of pollen protein exposure on the integrity of Caco-2 monolayer cells. (Changes in the activity of transwell external lactate dehydrogenase); Figure 7 This study demonstrates the effect of ricin exposure on the transmembrane resistance of Caco-2 monolayer cells. (TEER) 染毒后 / TEER 染毒前 ); Figure 8 This study demonstrates the effect of ricin exposure on the integrity of Caco-2 monolayer cells. (Concentration of fluorescein in the Transwell outer chamber); Figure 9 This study demonstrates the effect of ricin exposure on the integrity of Caco-2 monolayer cells. (Concentration of Evans blue in the Transwell outer chamber); Figure 10 This study demonstrates the effect of ricin exposure on the integrity of Caco-2 monolayer cells. (Changes in the activity of transwell external lactate dehydrogenase); Figure 11 This study demonstrates the effect of bovine serum albumin on the transmembrane resistance of Caco-2 monolayer cells. (TEER) 染毒后 / TEER 染毒前 ); Figure 12 This demonstrates the effect of bovine serum albumin on the integrity of Caco-2 monolayer cells. (Concentration of fluorescein in the Transwell outer chamber); Figure 13This demonstrates the effect of bovine serum albumin on the integrity of Caco-2 monolayer cells. (Concentration of Evans blue in the Transwell outer chamber); Figure 14 This demonstrates the effect of bovine serum albumin on the integrity of Caco-2 monolayer cells. (Changes in the activity of transwell external lactate dehydrogenase); Figure 15 This study demonstrates the effect of TBPH exposure on the transmembrane resistance of Caco-2 monolayer cells. (TEER) 染毒后 / TEER 染毒前 ); Figure 16 This demonstrates the effect of TBPH exposure on the integrity of Caco-2 monolayers. (Concentration of fluorescein in the Transwell outer chamber); Figure 17 This demonstrates the effect of TBPH exposure on the integrity of Caco-2 monolayers. (Concentration of Evans blue in the Transwell outer chamber); Figure 18 This demonstrates the effect of TBPH exposure on the integrity of Caco-2 monolayers. (Changes in the activity of transwell external lactate dehydrogenase); Figure 19 This demonstrates the effect of CdCl2 exposure on the transmembrane resistance of Caco-2 monolayer cells. (TEER) 染毒后 / TEER 染毒前 ); Figure 20 This study demonstrates the effect of CdCl2 exposure on the integrity of Caco-2 monolayer cells. (Concentration of fluorescein in the Transwell outer chamber); Figure 21 This study demonstrates the effect of CdCl2 exposure on the integrity of Caco-2 monolayer cells. (Concentration of Evans blue in the Transwell outer chamber); Figure 22 This study demonstrates the effect of CdCl2 exposure on the integrity of Caco-2 monolayer cells. (Changes in the activity of Transwell's outer chamber lactate dehydrogenase).

[0023] Figures 3-22 In the text, ** indicates that the P-value is less than 0.01, and * indicates that the P-value is less than 0.05. Detailed Implementation

[0024] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0025] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0026] In this application, the concentration of fluorescein in the lower chamber of the Transwell in the toxic group... 染毒后 The increase compared to the negative control group indicates that the integrity of the Caco-2 monolayer is disrupted and cell permeability is increased.

[0027] In this application, the concentration of Evans blue in the lower chamber of the Transwell in the toxic group was... 染毒后 The increase compared to the negative control group indicates that the integrity of the Caco-2 monolayer is disrupted and cell permeability is increased.

[0028] In this application, the change in lactate dehydrogenase activity (OD) is described. 染毒后 / OD 染毒前 A ratio of lactate dehydrogenase in the lower chamber of the Transwell equal to or greater than 1.15 indicates disruption of cell membrane integrity, increased cell membrane permeability, and leakage of intracellular lactate dehydrogenase into the lower chamber of the Transwell. Alternatively, statistical analysis can be used to determine the OD ratio. 染毒后 / OD 染毒前 The increase compared to the negative control group indicates that cell integrity has been compromised.

[0029] This application provides a method for evaluating the acute gastrointestinal toxicity of a test substance based on a Caco-2 cell model, characterized by comprising the following steps: Obtain Caco-2 monolayer cells; The Caco-2 monolayer cells are brought into contact with the test substance; The amount of fluorescent yellow permeating through the Caco-2 monolayer cells before and after contact was detected; and / or the amount of Evans blue permeating through the Caco-2 monolayer cells before and after contact was detected; and / or the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact was detected. The acute gastrointestinal toxicity of the test substance is evaluated based on the test results.

[0030] In some embodiments of this application, the amount of fluorescent yellow permeating the Caco-2 monolayer cells before and after contact and the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact are detected, and the acute gastrointestinal toxicity of the test substance is evaluated based on the detection results.

[0031] In some embodiments of this application, the amount of Evans blue permeating the Caco-2 monolayer cells before and after contact and the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact are detected, and the acute gastrointestinal toxicity of the test substance is evaluated based on the detection results.

[0032] In some embodiments of this application, evaluating the acute gastrointestinal toxicity of the test substance based on the test results includes the following: calculating the concentration of fluorescein. 染毒后 Evans Blue Concentration 染毒后 Changes in lactate dehydrogenase activity (OD) 染毒后 / OD 染毒前 Evaluation of acute gastrointestinal toxicity: Acute gastrointestinal toxicity is indicated by the presence of one or two of the following: OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of fluorescein in the exposed group 染毒后 Higher than the negative control group; or, OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of Evans blue in the treated group 染毒后 The results were higher than those of the negative control group; the negative control group was the result when the concentration of the test substance was 0.

[0033] In some embodiments of this application, evaluating the acute gastrointestinal toxicity of the test substance based on the test results includes the following: calculating the concentration of fluorescein. 染毒后 Evans Blue Concentration 染毒后 Changes in lactate dehydrogenase activity (OD) 染毒后 / OD 染毒前 Evaluation of acute gastrointestinal toxicity: Acute gastrointestinal toxicity is indicated by the presence of one or two of the following: OD 染毒后 / OD 染毒前 And / or the concentration of fluorescent yellow in the poisoned group 染毒后 All were higher than the negative control group; or, OD 染毒后 / OD 染毒前 And / or the concentration of Evans blue in the infected group 染毒后 All were higher than the negative control group; the negative control group was the result when the concentration of the test substance was 0.

[0034] In some embodiments of this application, the Caco-2 monolayer cells are cultured for at least 21 days before being brought into contact with the test substance; preferably, 21-28 days. For example, the culture time of Caco-2 cells can be 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or any range thereof.

[0035] In some embodiments of this application, the contact time between the Caco-2 monolayer cells and the test substance is less than 10 hours; preferably 1-6 hours. For example, the treatment time can be any range from 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 0.9 hours, 0.8 hours, 0.7 hours, 0.6 hours, 0.5 hours, 0.4 hours, 0.3 hours, 0.2 hours, 0.1 hours, or any time range thereof.

[0036] In some embodiments of this application, the test substance is selected from protein substances, chemicals, drugs, or food additives.

[0037] In some embodiments of this application, the protein is selected from one or both of pollen protein and ricin.

[0038] In some embodiments of this application, the concentration of the protein-like substance is 0.01-50 μg / ml. For example, the concentration of the protein-like substance can be 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.6 μg / ml, 0.8 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, 8 μg / ml, 10 μg / ml, 12 μg / ml, 14 μg / ml, or 16 μg / ml. 18 μg / ml, 20 μg / ml, 22 μg / ml, 24 μg / ml, 26 μg / ml, 28 μg / ml, 30 μg / ml, 32 μg / ml, 34 μg / ml, 36 μg / ml, 38 μg / ml, 40 μg / ml, 42 μg / ml, 44 μg / ml, 46 μg / ml, 48 μg / ml, 50 μg / ml or any range between them.

[0039] In some embodiments of this application, when the protein is pollen protein, the concentration of pollen protein is 0.1-50 μg / ml. The concentration of trichosanthes pollen protein can be 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.6 μg / ml, 0.8 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, 8 μg / ml, 10 μg / ml, 12 μg / ml, 14 μg / ml, 16 μg / ml, 18 μg / ml, 20 μg / ml, 22 μg / ml, 24 μg / ml, 26 μg / ml, 28 μg / ml, 30 μg / ml, 32 μg / ml, 34 μg / ml, 36 μg / ml, 38 μg / ml, 40 μg / ml, 42 μg / ml, 44 μg / ml, 46 μg / ml, 48 μg / ml, 50 μg / ml, or any range thereof.

[0040] In some embodiments of this application, when the protein is ricin, the concentration of ricin is 0.01-20 μg / ml. The concentration of ricin can be any range from 0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.6 μg / ml, 0.8 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, 8 μg / ml, 10 μg / ml, 12 μg / ml, 14 μg / ml, 16 μg / ml, 18 μg / ml, 20 μg / ml, or any range therebetween.

[0041] In some embodiments of this application, the chemical is selected from one or both of 2,3,4,5-tetrabromo-benzenediacarboxylic acid bis(2-ethylhexyl) ester (TBPH) and cadmium chloride (CdCl2).

[0042] In some embodiments of this application, the concentration of the chemical is 1-160000 nmol / L.

[0043] In some embodiments of this application, when the chemical is bis(2-ethylhexyl) 2,3,4,5-tetrabromo-benzenediacarboxylic acid (TBPH), the concentration of TBPH is 1-5000 nmol / L, preferably 1-2000 nmol / L; for example, the concentration of TBPH can be 1 nmol / L, 5 nmol / L, 10 nmol / L, 50 nmol / L, 100 nmol / L, 150 nmol / L, 200 nmol / L, 250 nmol / L, 300 nmol / L, 350 nmol / L, 400 nmol / L, 450 nmol / L, 500 nmol / L, 550 nmol / L, 600 nmol / L, 650 nmol / L, 7 00 nmol / L, 750 nmol / L, 800 nmol / L, 850 nmol / L, 900 nmol / L, 950 nmol / L, 1000 nmol / L, 1100 nmol / L, 1200 nmol / L, 1300 nmol / L, 1400 nmol / L, 1500 nmol / L, 1600 nmol / L, 1700 nmol / L, 1800 nmol / L, 1900 nmol / L, 2000 nmol / L, 2500 nmol / L, 3000 nmol / L, 3500 nmol / L, 4000 nmol / L, 4500 nmol / L, 5000 nmol / L, or any range between them.

[0044] In some embodiments of this application, when the chemical is cadmium chloride (CdCl2), the concentration of cadmium chloride is 0.01 μM-160 μM, preferably 10 μM-160 μM; the concentration of cadmium chloride can be 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM or any range thereof.

[0045] In some embodiments of this application, the method further includes a step of setting a positive control, wherein the positive control is 1% Triton-X 100.

[0046] In some embodiments of this application, the Caco-2 monolayer cells are washed with PBS or HBSS buffer before and after contact with the test substance.

[0047] This application provides the application of the above-mentioned acute gastrointestinal toxicity evaluation method in evaluating the gastrointestinal toxicity of drugs, food additives or chemicals.

[0048] Example 1. Materials: 1. Cells: Caco-2 cells were purchased from Beijing Baicaotai Biotechnology Co., Ltd.; DMEM culture medium containing 20% ​​fetal bovine serum (complete culture medium for Caco-2 cells, Wuhan Shangen Biotechnology Co., Ltd., catalog number: SNLM-068, batch number: SNLM202405414-2), Trichosanthes kirilowii pollen (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: B26236-1g, batch number: A21lB213311), and ricin (Beijing Feimo Biotechnology Co., Ltd., catalog number: FH81120-1mg, batch number: 202403) were used. 03), 2,3,4,5-Tetrabromo-benzenediacarboxylic acid bis(diethylhexyl) ester (TBPH) (Beijing Solarbio Science & Technology Co., Ltd., catalog number: SB7260), CdCl2 (Sinopharm Chemical Reagent Co., Ltd., batch number: 20230420), lactate dehydrogenase cytotoxicity assay kit (Beijing Regen Biotechnology Co., Ltd., catalog number: CT0027, batch number: 0205A25), Evans blue (Shandong Keyuan Biochemical Co., Ltd., catalog number: E808783-1g, batch number: C16223838), fluorescent yellow (thermo Fisher (China) Co., LTD, catalog number: L11252.06, batch number: 10233625).

[0049] Note: Trichosanthes kirilowii pollen protein was prepared in-house: 1.0 g of sample powder was placed in 5 mL of 0.1 mol / L KCl solution and soaked in a 4°C refrigerator for 24 h. Centrifuged at 4000 r / min for 20 min and collected the supernatant. Twice the volume of acetone was added, and the mixture was allowed to stand at low temperature for 1 h until the acetone evaporated. Centrifuged at 4000 r / min for 20 min and discarded the supernatant. The precipitate was collected, dissolved in 1 mL of PBS solution, centrifuged at 1000 r / min for 5 min, and the supernatant was collected. This is the trichosanthes kirilowii pollen protein extract. The molecular weight of the extract was analyzed by SDS gel electrophoresis to determine the extracted protein product. The protein content was then determined using the Coomassie Brilliant Blue method. The extract was then aliquoted and stored at -80°C for later use.

[0050] 2. Instruments: Fluorescence spectrophotometer (Shimadzu RF-6000), multi-functional microplate reader (FLUOstar Omega BMGLabtech), electric thermostatic incubator (ZDP-A2160A, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), carbon dioxide incubator (Alphavita BIO SCIENTIFIC, MCO-170-SR, Bingshan Songyang Biotechnology (Dalian) Co., Ltd.), cell resistance meter (Millicell® ERS-2, USAEMD Millipore Corporation).

[0051] 2. Test Procedure and Testing Indicators: The test procedure is as follows Figure 1 As shown.

[0052] Detection indicators: 1. Changes in transmembrane resistance of monolayer cells (TEER) 染毒后 / TEER 染毒前 ); 2. Fluorescent yellow permeability test; 3. Evans blue permeability test; 4. Detection of lactate dehydrogenase activity (OD) 染毒后 / OD 染毒前 ).

[0053] 3. Method: 3.1. Caco-2 monolayer cell culture like Figure 2 As shown, Caco-2 cells were seeded in DMEM culture medium (containing 20% ​​fetal bovine serum, 37°C, 5% CO2, and 90% humidity). The cells were seeded at a rate of 1×10⁻⁶ cells / day. 5 Seeds were placed at a density of 0.4 ml / well in the upper chamber (AP side) of the Transwell cell culture dish, as follows: Figure 1 As shown, 1 ml of culture medium was added to the lower chamber (BL side) of the petri dish. The culture medium was changed 24 hours after inoculation, then every other day for the next week, and then daily thereafter; the culture was used for experiments after day 21 of incubation.

[0054] 3.2. Treatment of Caco-2 cells before exposure to the virus 3.2.1 Measurement of transmembrane resistance (TEER) of monolayer cells Cells cultured for 21 days were washed three times with PBS and the lower chamber of a Transwell apparatus before adding the test substance. Then, 0.4 ml of complete culture medium was added to the upper chamber and 1 ml to the lower chamber. The mixture was incubated at room temperature for 0.5 h to equilibrate, and then the transmembrane resistance of the monolayer was measured. The method is as follows: [The text abruptly ends here, likely due to an incomplete translation or missing information.] ® On the ERS-2 cell resistance meter, select the detection resistance, turn on the power switch, and vertically insert the electrodes into the upper and lower chambers of the Transwell. Once the readings stabilize, record the resistance before exposure. Measure three different points on each plate.

[0055] 3.2.2 Fluorescent Yellow and Evans Blue Penetration Test Before adding the test substance, the cells cultured for 21 days were washed with PBS and placed in the Transwell lower chamber three times.

[0056] 3.2.3 Detection of lactate dehydrogenase activity Before adding the test substance, cells cultured for 21 days were washed three times with PBS and the lower chamber of the Transwell apparatus. 1 ml of complete culture medium was added to the lower chamber of the Transwell apparatus, and 0.4 ml of complete culture medium was added to the upper chamber. The mixture was incubated at room temperature for 0.5 h to equilibrate. 5 μl of culture medium was then aspirated from the lower chamber of the Transwell apparatus, and lactate dehydrogenase activity was measured according to the method described in the lactate dehydrogenase cytotoxicity assay kit. Three replicates were performed per blank.

[0057] 3.2.4 Poisoning Cells were individually exposed to the test substances. 0.4 ml of the test substance was added to the upper chamber of the Transwell, and 1 ml of culture medium was added to the lower chamber. Exposure was repeated for 1 h, 3 h, and 6 h. The test substances were: pollen protein (concentrations of 0 μg / ml (negative control), 0.625 μg / ml, 1.25 μg / ml, 8 μg / ml, and 40 μg / ml, and positive control), ricin (concentrations of 0 μg / ml (negative control), 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, and positive control), and bovine serum albumin (concentration of 0 mg / ml). The concentrations of Triton-X 100 were: negative control group (6.25 mg / ml, 12.5 mg / ml, 25 mg / ml and 50 mg / ml and positive control), TBPH (TBPH concentrations: 0 nmol / L (negative control group), 1 nmol / L, 10 nmol / L, 100 nmol / L, 1000 nmol / L and positive control), and CdCl2 (CdCl2 concentrations: 0 μM (negative control group), 20 μM, 40 μM, 80 μM, 160 μM and positive control). The positive control was 1% Triton-X 100.

[0058] 4. Testing of various indicators after the end of drug use. 4.1 Measurement of transmembrane resistance (TEER) of monolayer cells At the end of the treatment, the cells and the lower chamber of the Transwell were gently washed three times with PBS. 1 ml of complete culture medium was added to the lower chamber, and 0.4 ml of complete culture medium was added to the upper chamber. The cells were incubated at room temperature for 0.5 h to equilibrate, and then the transmembrane resistance of the monolayer was measured. The method is as follows: In Millicell... ® On the ERS-2 cell resistance meter, select the detection resistance, turn on the power switch, and vertically insert the electrodes into the upper and lower chambers of the Transwell. Once the readings stabilize, record the resistance after exposure. Measure three different points on each plate.

[0059] 4.2 Fluorescent Yellow Penetration Test At the end of the treatment, the cells and the lower chamber of the Transwell membrane were gently washed three times with HBSS buffer pre-warmed to 37°C. 0.4 ml of fluorescein (250 μg / ml) solution was added to the upper chamber of the Transwell membrane, followed by 0.6 ml of blank HBSS solution in the lower chamber. The membrane was then incubated at 37°C in a 5% CO2 cell culture incubator for 1 hour. 0.5 ml of the solution was then removed from the lower chamber, and its absorbance (OD value) was measured using a fluorescence spectrophotometer at an excitation wavelength of 420 nm and an emission wavelength of 545 nm. The concentration of fluorescein in the lower chamber was calculated using a standard curve of fluorescein concentration versus OD value.

[0060] Fluorescein concentration-OD value standard curve: Prepare fluorescein solutions with concentrations of 0, 0.01, 0.1, 1, and 10 μg / ml. Weigh 1 mg of fluorescein dye, dissolve it in HBSS solution, mix thoroughly, and bring the volume to 10 ml. Add 0.5 ml of a 100 μg / ml fluorescein solution to 4.5 ml of HBSS solution to prepare a 10 μg / ml fluorescein solution. Add 0.5 ml of the 10 μg / ml fluorescein solution to 4.5 ml of HBSS solution to prepare a 1 μg / ml fluorescein solution. Add 0.5 ml of the 1 μg / ml fluorescein solution to 4.5 ml of HBSS solution to prepare a 0.1 μg / ml solution. Add 0.5 ml of the 0.1 μg / ml fluorescein solution to 4.5 ml of HBSS solution to prepare a 0.01 μg / ml solution. A fluorescence spectrophotometer was used to measure the absorbance (OD value) of fluorescein solutions with concentrations of 0.01, 0.1, 1, and 10 μg / ml, with HBSS solution as a blank and zeroing at an excitation wavelength of 420 nm and an emission wavelength of 545 nm. A standard curve of fluorescein concentration versus OD value was plotted.

[0061] 4.3 Evans Blue Penetration Test At the end of the treatment, the cells and the lower chamber of the Transwell were gently washed three times with PBS buffer pre-warmed to 37°C. 1000 μL of 4% BSA solution was added to the lower chamber, and 400 μL of EB-BSA working solution (4% EB stock solution mixed with 60 volumes of 4% BSA, final concentration 0.67 mg / mL) was added to the upper chamber to ensure consistent liquid levels and prevent leakage caused by height differences. The cells were incubated at 37°C in a 5% CO2 cell culture incubator for 1 hour. The liquid from the lower chamber was then transferred to a 1.5 mL EP tube, centrifuged at 1000 rpm for 5 minutes, and 200 μL was added to a 96-well plate. The absorbance (OD value) was measured at 620 nm using a multi-plate reader. The concentration of Evans blue in the lower chamber was calculated based on the Evans blue concentration-OD value standard curve.

[0062] Evans blue concentration-OD value standard curve: Evans blue solutions with concentrations of 0, 0.512, 2.56, 12.8, and 64 μg / ml were prepared. Weigh 0.32 mg of Evans blue dye, dissolve it in 4% BSA solution, mix thoroughly, and dilute to 5 ml to prepare an Evans blue solution with a concentration of 64 μg / ml. Then, add 1 ml of the 64 μg / ml Evans blue solution to 4 ml of 4% BSA solution, mix well, and prepare an Evans blue solution with a concentration of 12.8 μg / ml. Next, add 1 ml of the 12.8 μg / ml Evans blue solution to 4 ml of 4% BSA solution, mix well, and prepare an Evans blue solution with a concentration of 2.56 μg / ml. Finally, add 1 ml of the 2.56 μg / ml Evans blue solution to 4 ml of 4% BSA solution, mix well, and prepare an Evans blue solution with a concentration of 0.512 μg / ml. A multi-functional microplate reader was used at a wavelength of 620 nm, with 4% BSA as the blank solution for zeroing. The absorbance (OD) of Evans blue solutions at concentrations of 0.512, 2.56, 12.8, and 64 μg / ml was measured. A standard curve of Evans blue concentration versus OD value was plotted.

[0063] 4.4 Detection of lactate dehydrogenase activity At the end of the exposure, 5 μL of culture medium was aspirated from the lower chamber of the Transwell assay, and lactate dehydrogenase activity was measured according to the method described in the lactate dehydrogenase cytotoxicity assay kit. Three replicates were performed per well.

[0064] Take 5 μL of Transwell lower chamber culture medium, add 25 μL of LDH buffer and 5 μL of NAD buffer respectively, mix well, and incubate at 37℃ for 15 min; then add 25 μL of phenylhydrazine chromogenic solution, mix well, and incubate at 37℃ for 15 min; then add 100 μL of alkaline chromogenic solution and 150 μL of ultrapure water in sequence, mix well, and let stand at room temperature for 5 min. Measure the absorbance of each well at 440 nm using a multi-functional microplate reader.

[0065] 5. Data processing for various testing indicators 5.1 Measurement of transmembrane resistance (TEER) of monolayer cells: The resistance of monolayer cells after exposure to the toxin was compared with that before exposure, i.e., TEER was calculated. 染毒后 / TEER 染毒前 The ratios were then analyzed statistically. Changes in electrical resistance before and after exposure were compared between different doses.

[0066] 5.2 Fluorescein Permeation Test: Based on the standard curve of fluorescein concentration-OD value, the concentration of fluorescein in the lower chamber was calculated, and then corresponding statistical analysis was performed. The changes in fluorescein concentration between each dose group and the solvent control group were compared.

[0067] 5.3 Evans Blue Permeation Test: Based on the standard curve of Evans blue concentration-OD value, the concentration of Evans blue in the lower chamber was calculated, and then corresponding statistical analysis was performed. The changes in Evans blue concentration between each dose group and the solvent control group were compared.

[0068] 5.4 Detection of lactate dehydrogenase activity: The OD value of lactate dehydrogenase after exposure in the lower chamber is compared with the OD value of lactate dehydrogenase before exposure, i.e., the OD value is calculated. 染毒后 / OD 染毒前 The ratios were then analyzed statistically. Changes in LDH activity before and after exposure to different doses were compared.

[0069] result: 1. Changes in transmembrane resistance, permeability, and lactate dehydrogenase activity of Caco-2 monolayer cells at different exposure times to pollen protein. One hour after exposure to pollen protein, the concentration of fluorescein in the transwell outer chamber of cells in the 8 μg / ml dose group significantly increased. Six hours after exposure, the concentration of fluorescein in the transwell outer chamber of cells in the 0.625 μg / ml dose group significantly increased. Three hours after exposure, the concentration of Evans blue in the transwell outer chamber of cells in the 0.625 μg / ml dose group increased. The transmembrane resistance of Caco-2 monolayer cells significantly decreased at 40 μg / ml (high dose group) at 6 hours after exposure, indicating that the cell membrane integrity of Caco-2 monolayer cells was damaged. No significant changes were observed in the transwell outer chamber lactate dehydrogenase activity of cells in each pollen protein exposure group at 1, 3, and 6 hours after exposure.

[0070] Figures 3-6The experimental results showed that, among the methods for detecting cell membrane integrity after Caco-2 monolayer cells were exposed to pollen protein, the fluorescent yellow permeation assay was the most sensitive, followed by the Evans blue permeation assay. Changes in the transmembrane resistance of Caco-2 monolayer cells appeared relatively late, while changes in lactate dehydrogenase activity were not observed.

[0071] 2. Changes in transmembrane resistance, permeability, and lactate dehydrogenase activity of Caco-2 monolayer cells at different exposure times to ricin. One hour after ricin exposure, the concentrations of fluorescein, Evans blue, and lactate dehydrogenase activity in the transwell outer chamber all significantly increased. Increased Evans blue concentration was observed in the transwell outer chamber at a dose of 0.1 μg / ml, while the doses showing increased fluorescein concentration and lactate dehydrogenase activity were both 1 μg / ml. Transmembrane resistance in Caco-2 monolayer cells significantly decreased at 3 and 6 hours post-exposure in both the 1 μg / ml and 10 μg / ml dose groups, indicating damage to the cell membrane integrity of Caco-2 monolayer cells.

[0072] Figures 7-10 The experimental results showed that, among the methods for detecting cell membrane integrity in Caco-2 monolayer cells exposed to ricin, the Evans blue permeation test was the most sensitive, followed by the fluorescent yellow permeation test and changes in lactate dehydrogenase activity, while changes in transmembrane resistance of Caco-2 monolayer cells appeared relatively late.

[0073] 3. Changes in transmembrane resistance, permeability, and lactate dehydrogenase activity of Caco-2 monolayer cells after different treatment times with bovine serum albumin. Figures 11-14 The results showed that no changes in transmembrane resistance, fluorescein concentration in the transwell outer chamber, Evans blue concentration, or lactate dehydrogenase activity were observed at any time point and in any dose group after bovine serum albumin (BSAL) exposure. This indicates that BSAL does not cause any alteration in the cell membrane integrity of Caco-2 monolayers.

[0074] 4. Changes in transmembrane resistance, permeability, and lactate dehydrogenase activity of Caco-2 monolayer cells at different exposure times to bis(diethylhexyl) 2-bromo-phenylenedicarboxylic acid (TBPH). Figures 15-18The results showed that after Caco-2 monolayer cells were treated with 2,3,4,5-tetrabromo-benzenediacarboxylic acid bis(diethylhexyl) ester (TBPH), increased lactate dehydrogenase activity was observed in the transwell outer chamber of cells at 3 h post-treatment (1000 nmol dose group); and at 6 h post-treatment (100 nmol dose group), increased lactate dehydrogenase activity was observed in the transwell outer chamber. No changes in transmembrane resistance, concentrations of fluorescein and Evans blue in the transwell outer chamber were observed at any time point or in any dose group.

[0075] 5. Changes in transmembrane resistance, permeability, and lactate dehydrogenase activity of Caco-2 monolayer cells at different CdCl2 exposure times. One hour after Caco-2 monolayer cells were exposed to CdCl2, changes in transmembrane resistance, concentration of fluorescein in the transwell outer chamber, concentration of Evans blue, and activity of lactate dehydrogenase could be detected.

[0076] Figures 19-22 The results showed that in the 20 μM dose group, increased lactate dehydrogenase activity was observed; in the 40 μM dose group, increased fluorescein concentration and transmembrane resistance were observed; and in the 80 μM dose group, increased Evans blue concentration was observed. With increasing dose or exposure time, the concentrations of fluorescein, Evans blue, and lactate dehydrogenase activity in the transwell outer chamber all increased. Regarding the change in transmembrane resistance of Caco-2 monolayer cells, at 1 hour of exposure, the resistance initially increased and then decreased with increasing dose, and gradually decreased with prolonged exposure time.

[0077] Table 1 lists the changes in lactate dehydrogenase activity (OD) in the negative control group after different exposure times for different test substances. 染毒后 / OD 染毒前 )

[0078] Note: a represents the lactate dehydrogenase activity (OD) of the negative control group with different exposure times to different test substances. 染毒后 / OD 染毒前 All values ​​are averages (n=3); b represents the lactate dehydrogenase activity (OD) of the negative control group with different exposure times to different test substances. 染毒后 / OD 染毒前 The mean is the average of the mean ± SD; c is the mean ± 1.96 SD, which is the normal range calculated.

[0079] As shown in Table 1, the normal range for lactate dehydrogenase activity is 96.9-115.1. A value exceeding 115.1 indicates poisoning. Therefore, OD...染毒后 / OD 染毒前 A ratio greater than or equal to 1.15 indicates that the integrity of the cell membrane is compromised, cell membrane permeability is increased, and the cell is toxic.

[0080] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Anyone skilled in the art may make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent application.

Claims

1. A method for evaluating the acute gastrointestinal toxicity of a test substance based on a Caco-2 cell model, characterized in that, Includes the following steps: Obtain Caco-2 monolayer cells; The Caco-2 monolayer cells are brought into contact with the test substance; The amount of fluorescent yellow permeating through the Caco-2 monolayer cells before and after contact was detected; and / or the amount of Evans blue permeating through the Caco-2 monolayer cells before and after contact was detected; and / or the change in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact was detected. The acute gastrointestinal toxicity of the test substance is evaluated based on the test results.

2. The method according to claim 1, wherein, The amount of fluorescent yellow permeating the Caco-2 monolayer cells before and after contact was detected, and the changes in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact were detected; or The amount of Evans blue permeating the Caco-2 monolayer cells before and after contact was detected, as well as the changes in lactate dehydrogenase activity in the external culture medium of the Caco-2 monolayer cells before and after contact.

3. The method according to claim 1, wherein, The evaluation of acute gastrointestinal toxicity of test substances based on test results includes the following: Calculate the concentration of fluorescein 染毒后 Evans Blue Concentration 染毒后 Changes in lactate dehydrogenase activity (OD) 染毒后 / OD 染毒前 ; Assessing acute gastrointestinal toxicity: Acute gastrointestinal toxicity is indicated by the presence of one or two of the following: OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of fluorescein in the exposed group 染毒后 Higher than the negative control group; or, OD 染毒后 / OD 染毒前 ≥115%, and / or the concentration of Evans blue in the treated group 染毒后 Higher than the negative control group; or, OD 染毒后 / OD 染毒前 And / or the concentration of fluorescent yellow in the poisoned group 染毒后 All were higher than the negative control group; or, OD 染毒后 / OD 染毒前 And / or the concentration of Evans blue in the infected group 染毒后 All were higher than the negative control group; The negative control group is the result when the concentration of the test substance is 0.

4. The method according to claim 1, wherein, The contact time between the Caco-2 monolayer cells and the test substance is less than 10 hours; preferably 1-6 hours.

5. The method according to claim 1, wherein, Before the Caco-2 monolayer cells are brought into contact with the test substance, the Caco-2 cells are cultured for at least 21 days; preferably 21-28 days.

6. The method according to claim 1, wherein, The test substance is selected from protein substances, chemicals, drugs, or food additives.

7. The method according to claim 6, wherein, The protein is selected from one or both of pollen protein and ricin.

8. The method according to claim 7, wherein, The concentration of protein substances is 0.01-50 μg / ml.

9. The method according to claim 8, wherein, When the protein is pollen protein, the concentration of pollen protein is 0.1-50 μg / ml; When the protein is ricin, the concentration of ricin is 0.01-20 μg / ml.

10. The method according to any one of claims 1 to 9, wherein, The chemical is selected from one or both of 2,3,4,5-tetrabromo-benzenediacarboxylic acid bis(2-ethylhexyl) ester (TBPH) and cadmium chloride (CdCl2).

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