Application of danshenolic acid in reducing cytotoxicity of rat liver S9 cells

Abstract

The present invention discloses the application of danshenolic acid in reducing the cytotoxicity of rat liver S9 cells. The present invention for the first time discovers that danshenolic acid has a strong protective effect on the cytotoxicity of the chemical mutagenicity test of rat liver S9 added with an in vitro activator, and does not affect the detection of mutagenicity. Danshenolic acid can be used as a protective agent for reducing the cytotoxicity of rat liver S9 cells; the present invention also provides a culture method for the mutagenicity test of reducing the cytotoxicity of rat liver S9 cells, which is economical and easily available; it is applicable to the mutagenicity test of adherent cells cultured in vitro in the safety evaluation of industrial chemicals, drugs and environmental chemicals.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and particularly relates to the application of danshenolic acid in reducing the cytotoxicity of rat liver S9 cells. Background Art

[0002] In safety evaluation tests such as mutagenicity and carcinogenicity of chemical substances, a considerable part of chemical substances need to be metabolically activated to cause mutagenicity and carcinogenicity. Therefore, in the non-clinical safety evaluation of new drugs or new chemical substances, an appropriate activation system should be used in short-term in vitro tests. Rat liver S9 is the mitochondrial-free supernatant of rat liver tissue homogenate, which contains a large number of chemical substance-metabolizing enzymes and is often used as a biotransformation activation system for in vitro tests. Conventional liver S9 is obtained from rats given metabolic enzyme inducers. Rat liver S9 can be used at a relatively high dose in the bacterial reverse mutation test, but it shows certain cytotoxicity in the in vitro cultured cell mutagenicity test, which limits the dosage used. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide the application of danshenolic acid.

[0004] The purpose of the second aspect of the present invention is to provide a product.

[0005] The purpose of the third aspect of the present invention is to provide a mutagenicity test method.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided the application of danshenolic acid in reducing the toxicity of the metabolic activation system and / or preparing a product for reducing the toxicity of the metabolic activation system.

[0008] Preferably, the metabolic activation system includes a rat liver S9 mixture.

[0009] Preferably, the metabolic activation system is used for mutagenicity tests.

[0010] Preferably, the mutagenicity test includes a cell mutagenicity test.

[0011] Preferably, the cells include Chinese hamster lung cell lines (CHL, V79), Chinese hamster ovary (CHO) cells.

[0012] Preferably, the cell mutagenicity test includes chemical substance mutagenicity.

[0013] Preferably, the chemical substances include cyclophosphamide and benzo[a]pyrene.

[0014] In the second aspect of the present invention, there is provided a product, and the product contains danshenolic acid.

[0015] Preferably, the product further contains rat liver S9 mixture.

[0016] Preferably, the product further contains matrix components.

[0017] Preferably, the matrix components include N-cyclohexyl-N'-(2-morpholinoethyl)aminocyanomethanesulfonate and type I rat tail collagen.

[0018] Preferably, the product contains a carrier or excipient.

[0019] Preferably, the product is a drug or reagent.

[0020] In the third aspect of the present invention, a mutagenicity test method is provided, which includes the step of using the product described in the second aspect of the present invention.

[0021] Preferably, the mutagenicity test includes a cell mutagenicity test.

[0022] Preferably, the cells include Chinese hamster lung cell lines (CHL, V79), Chinese hamster ovary (CHO) cells, and peripheral blood lymphocytes.

[0023] Preferably, the mutagenicity includes chemical mutagenicity.

[0024] Preferably, the chemicals include cyclophosphamide and benzo[a]pyrene.

[0025] Preferably, the mutagenicity test includes the following steps:

[0026] S1: Add danshenolic acid to the rat tail collagen solution, and then add it to the cell culture plate to complete the coating of the cell culture plate;

[0027] S2: Inoculate cells, add chemicals and rat liver S9 mixture, and after culturing for 4 - 8 h, replace the culture medium without chemicals and S9 mixture for culturing.

[0028] Preferably, the concentration of type I rat tail collagen is 80 - 120 μg / mL.

[0029] Preferably, the type I rat tail collagen solution is prepared from N-cyclohexyl-N'-(2-morpholinoethyl)aminocyanomethanesulfonate solution.

[0030] Preferably, the concentration of N-cyclohexyl-N'-(2-morpholinoethyl)aminocyanomethanesulfonate is 100 - 160 μg / mL.

[0031] Preferably, the concentration of danshenolic acid is 30 - 50 μg / mL.

[0032] Preferably, the chemicals include cyclophosphamide and benzo[a]pyrene.

[0033] Preferably, the concentration of cyclophosphamide is 8 - 12 μg / mL.

[0034] Preferably, the concentration of benzo[a]pyrene is 4 - 6 μg / mL.

[0035] Preferably, the protein concentration in the rat liver S9 mixture is 5 - 15 mg / mL.

[0036] Preferably, the rat liver S9 mixture further contains 6 - 10 mmol / L MgCl2, 30 - 36 mmol / L KCl, 4 - 6 mmol / L glucose - 6 - phosphate, and 3 - 5 mmol / L coenzyme II.

[0037] The beneficial effects of the present invention are as follows:

[0038] The present invention firstly discovers that danshenolic acid has a strong protective effect on the cytotoxicity of the chemical mutagenicity test of rat liver S9 added with an in vitro activator, and does not affect the detection of mutagenicity. Danshenolic acid can be used as a protective agent to reduce the cytotoxicity of rat liver S9. The present invention also provides a culture method for the mutagenicity test of reducing the cytotoxicity of rat liver S9 cells, which is economical and easily available; it is applicable to the mutagenicity test of adherent cells in vitro culture in the safety evaluation of industrial chemicals, drugs, and environmental chemicals. Description of the Drawings

[0039] Figure 1 It is the application result of the influence of the culture method of the present invention and the conventional culture method on the CHL cytotoxicity of rat liver S9.

[0040] Figure 2 It is the result of the CHL cell chromosome aberration test (adding rat liver S9) of the culture method of the present invention and the conventional culture method. Detailed Embodiments

[0041] The following will clearly and completely describe the concept and the technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0042] The chemical reagents used in the present invention are: thiazolyl blue, cyclophosphamide, benzo[a]pyrene, colchicine, danshenolic acid, N - cyclohexyl - N′-(2 - morpholinoethyl)aminocarbocyanine p - toluenesulfonate. The above chemicals are all purchased from SIGMA Company, and are analytical pure reagents.

[0043] The present invention establishes a culture method for reducing the cytotoxicity of rat liver S9 cells, which has a strong protective effect on the cytotoxicity of chemical substances in the cell mutagenicity test of rat liver S9 supplemented with an in vitro activator.

[0044] All cells used in this example are the Chinese hamster lung cell line CHL.

[0045] Example 1: Selection of the dose of the protective agent

[0046] 1. Adopt the toxicity evaluation method of the present invention

[0047] (a) Dilute type I rat tail collagen to 100 μg / mL with an aqueous solution of N-cyclohexyl-N'-(2-morpholinoethyl)aminocyanomethane p-toluenesulfonate with a concentration of 130 μg / mL;

[0048] (b) Add danrofenic acid at 4 concentrations of 0, 10, 20, 30, and 50 μg / mL to the rat tail collagen solution prepared in step (a), then add it to a 96-well cell culture plate, with 3 parallel wells for each concentration, and place it in an incubator at 37°C with a 5% carbon dioxide concentration for 3 - 4 h to complete the coating of the cell culture plate; then rinse with sterile water and phosphate buffer solution for later use; at the same time, set 6 uncoated wells, among which 3 wells are the negative control group and 3 wells are the conventional culture method group;

[0049] (c) Adjust the cell density of the Chinese hamster lung cell line CHL cells to a cell suspension of 1×10 5 cells / mL; inoculate the above cell suspension into the above-treated 96-well cell culture plate, 100 μL per well, and place it in an incubator at 37°C (5% CO2, >90% humidity) for 24 h;

[0050] (d) After the cells grow into a monolayer, aspirate the original culture medium, and add S9 mixture with a protein concentration of 10 mg / mL (8 mmol / L MgCl2, 33 mmol / L KCl, 5 mmol / L glucose-6-phosphate, 4 mmol / L coenzyme II) respectively. After culturing for 6 h, replace the culture medium without S9 and continue culturing for 18 h.

[0051] 2. Apply the conventional cell cytotoxicity test method, including the following steps: aspirate the original culture medium, add 50 μL of thiazolyl blue (MTT) solution (1 mg / mL) to each well, culture at 37°C with 5% CO2 for 2 hours, then discard the MTT solution, and add 100 μL of isopropanol solution to each well.

[0052] Shake the plate, measure the absorbance value at a wavelength of 570 nm (reference wavelength 650 nm), and calculate the cell survival rate.

[0053] The results of evaluating the cytotoxicity of rat liver S9 cells by the conventional method and the method of the present invention are shown inFigure 1 The results showed that at rosmarinic acid concentrations of 30 and 50 μg / mL, the culturing method of the present invention had a significant protective effect on the cytotoxicity of rat liver S9.

[0054] Example 2: Influence of the culturing method of the present invention on the mutagenic effect of positive substances

[0055] (a) Dilute type I rat tail collagen to 100 μg / mL with an aqueous solution of N-cyclohexyl-N'-(2-morpholinoethyl)aminocyanomethane p-toluenesulfonate at a concentration of 130 μg / mL.

[0056] (b) Add 30 μg / mL of rosmarinic acid to the rat tail collagen solution prepared in step (a), then add it to a cell culture flask with a bottom area of 25 cm 2 and place it at 37 °C in an environment with a carbon dioxide concentration of 5% for 3 - 4 h to complete the coating of the cell culture flask. Then rinse it with sterile water and phosphate buffer solution for later use.

[0057] (c) Three culture flasks were used for the culturing method of the present invention and the conventional method respectively. Digest the passage cells with 0.25% trypsin solution and inoculate 7.5×10 5 cells per flask into the culture flasks and culture for 24 h.

[0058] (d) Replace the culture medium (RPMI-1640 culture medium), add cyclophosphamide at a final concentration of 10 μg / mL or benzo[a]pyrene at 5 μg / mL, and add 0.4 mL of S9 mixture (8 mmol / L MgCl2, 33 mmol / L KCl, 5 mmol / L glucose-6-phosphate, 4 mmol / L coenzyme II) with a protein concentration of 10 mg / mL to each flask. After culturing for 6 h, replace the culture medium without the test substance and S9 and continue culturing for 18 h.

[0059] (e) 1 - 3 h before collecting the cells, observe the cell morphology and growth status. Add 0.15 mL of 0.01 mg / mL colchicine to each flask of cells. Digest the cells with 0.25% trypsin solution, transfer them to a centrifuge tube, and centrifuge at 800 rpm for 5 minutes. Hypotonicize with 0.075 mol KCl solution at 37 °C for 30 min and fix twice with methanol: glacial acetic acid (3:1). Drop the cells to prepare chromosome specimens and stain with Giemsa.

[0060] (f) Observe 100 metaphase cells in each culture flask, and observe and record the chromosome number and chromosomal structural aberrations respectively. At the same time, observe and record the polyploidy and endoreduplication that occur. The observation of chromosomal structural aberrations should include gaps (not counted as aberrations), breaks, chromatid and chromosomal fragments (acentric chromosomes or chromatid fragments, etc.), deletions, exchanges, rings, and minute bodies, etc. When one or more types of aberration occur in a cell, it is recorded as an aberrant cell. Calculate the aberration rate of cells in each group (the percentage of aberrant cells in the total observed cells). Cells with chromosome pulverization and severe damage (number of aberrations ≥ 10) should also be recorded.

[0061] Using the conventional method (refer to "GB / T 21794-2008 Test Method for Chromosomal Aberrations in vitro in Mammalian Cells by Chemicals") and the method of the present invention to evaluate the results of the chromosomal aberration rate of the mutagenic positive substances cyclophosphamide and benzo[a]pyrene on CHL cells are shown in Figure 2 , and the results show that the culture method for reducing the toxicity of rat liver S9 cells described in the present invention has no significant difference in the chromosomal aberration rate of CHL cells caused by 2 indirect mutagenic positive substances compared with the conventional method.

[0062] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of danshenolic acid in reducing the toxicity of metabolic activation systems in vitro and / or in preparing a product for reducing the toxicity of metabolic activation systems; characterized in that, The metabolic activation system is a rat liver S9 mixture; the concentration of danshenolic acid is 30-50 μg / mL.

2. The application according to claim 1, characterized in that The metabolic activation system is used for mutagenicity tests.

3. The application according to claim 2, characterized in that The mutagenicity test includes a cell mutagenicity test.

4. The application according to claim 3, characterized in that The cells include Chinese hamster lung cells or Chinese hamster ovary cells.

5. The application according to claim 3, characterized in that The cell mutagenicity test includes chemical mutagenesis.

6. The application according to claim 5, wherein The chemicals include cyclophosphamide or benzo[a]pyrene.

7. A culture method for a cell mutagenicity test that reduces the cytotoxicity of rat liver S9 cells, characterized in that, It includes the following steps: S1: Add danshenolic acid to the rat tail collagen solution, and then add it to the cell culture plate to complete the coating of the cell culture plate; S2: Inoculate cells, add chemicals and rat liver S9 mixture, and after culturing for 4-8 h, replace the culture medium without chemicals and S9 mixture for culture; The concentration of danshenolic acid is 30-50 μg / mL; The chemical is cyclophosphamide or benzo[a]pyrene.

8. The culturing method according to claim 7, characterized in that, The protein concentration in the rat liver S9 mixture is 8-12 mg / mL.

9. The culturing method according to claim 8, characterized in that, The rat liver S9 mixture also includes 6-10 mmol / L MgCl2, 30-36 mmol / L KCl, 4-6 mmol / L glucose-6-phosphate, and 3-5 mmol / L coenzyme II.

10. The cultivation method according to claim 7, wherein The concentration of cyclophosphamide is 8-12 μg / mL.

11. The cultivation method according to claim 7, wherein The concentration of benzo[a]pyrene is 4-6 μg / mL.

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