New application of ganoderic acid B

Abstract

The invention discloses novel application of ganoderic acid B, belongs to the field of liver injury treatment, and particularly relates to application of ganoderic acid B in preparation of a medicine for treating liver cell injury caused by aflatoxin. According to the invention, human liver cancer HepG2 cells are utilized to construct an AFB1 cell injury model, lucid ganoderma active ingredients with a protection effect on injury are screened out, and the resistance mechanism of the lucid ganoderma active ingredients is researched and predicted, so that a new method and a new target spot are provided for searching for diseases caused by AFB1 prevention and treatment. A new method can be provided for preventing and treating the liver cancer caused by the AFB1.

Description

Technical Field

[0001] The present invention belongs to the field of liver injury treatment, and particularly relates to a new use of ganoderic acid B. Background Art

[0002] Liver cancer refers to malignant tumors that occur in the liver, which can be divided into two major categories: primary liver cancer and secondary liver cancer. Among them, hepatocellular carcinoma accounts for 75-85% of primary liver cancer and is one of the most common malignant tumors clinically. Aflatoxin has high carcinogenicity and teratogenicity and mainly exists in foods such as mold-infected grains, nuts, and corn. Especially in tropical and subtropical regions, due to climate conditions and improper food preservation, the risk of aflatoxin contamination is relatively high, causing immeasurable losses to food and agricultural production. Moreover, studies have shown that in patients with hepatitis B or C virus, aflatoxin exposure further increases the risk of liver cancer in patients. Therefore, this has become a public health problem that cannot be ignored.

[0003] Aflatoxin belongs to the class of difuranocoumarin derivatives and is mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. Its molecular structure is quite stable, and it cannot be degraded by high-temperature treatment at 200 °C or ultraviolet irradiation. It has carcinogenic, teratogenic, mutagenic, hepatotoxic, and immunosuppressive effects. Among the more than 20 known aflatoxins, the most common and important ones are four types: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among them, AFB1 has the greatest toxicity and the strongest carcinogenicity, and the International Agency for Research on Cancer has classified it as a Group 1 carcinogen. The liver, as the main organ of human metabolism, mediates the detoxification of AFB1 and other mycotoxins, and is also the organ most vulnerable to AFB1.

[0004] Ganoderma lucidum is a medicinal fungus with various biological activities and has a history of more than two thousand years of medical use in China. Its main active ingredients include ganoderma polysaccharides, ganoderma triterpenoids, nucleosides, sterols, and proteins. Modern research has found that Ganoderma lucidum has anti-tumor, immunomodulatory, hypoglycemic, liver-protecting, anti-aging, antioxidant, and anti-inflammatory effects. At present, there are many studies on Ganoderma lucidum in the field of anti-tumor, and its main mechanisms of action involve regulating the body's immunity, blocking the cell cycle, inducing apoptosis and autophagy of tumor cells, inhibiting the metastasis of tumor cells, and antioxidant effects. However, there are no reports on its related research on resisting AFB1-induced DNA damage in cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a new use of ganoderic acid B.

[0006] The application of Ganoderic acid B of the present invention is in the preparation of a drug for treating hepatocyte damage caused by aflatoxin.

[0007] Further, the aflatoxin is aflatoxin B1.

[0008] Further, the hepatocyte damage is DNA damage.

[0009] Further, the molar concentration of Ganoderic acid B is 5 - 40 μmol / L.

[0010] The present invention uses human hepatoma HepG2 cells to construct an AFB1 cell damage model, screens out Ganoderma active ingredients with a protective effect on the damage, and studies and predicts its resistance mechanism, in order to provide new methods and new targets for finding the prevention and treatment of diseases caused by AFB1. It can be written as providing a new method for the prevention and treatment of liver cancer caused by AFB1. Brief Description of the Drawings

[0011] Figure 1 Results of treating human hepatoma HepG2 cells with different concentrations of AFB1; A: Effect of AFB1 treatment with concentration gradient on the viability of HepG2 cells, B: Effect of AFB1 treatment with concentration gradient on the content of γ-H2AX in HepG2 cells, C: Effect of time gradient treatment with 60 μmol / L AFB1 on the number of γ-H2AX foci in HepG2 cells;

[0012] Figure 2 Diagram for screening Ganoderma active ingredients with a protective effect on AFB1 damage; A: Effect of Ganoderic acid treatment with concentration gradient on the viability of HepG2 cells, B: Protective effect of pretreatment with Ganoderic acids A, B, C, D, F, J on AFB1 damage, C: Protective effect of pretreatment with different concentrations of Ganoderic acid B on AFB1 damage (*p<0.05; **p<0.01; ***p<0.001);

[0013] Figure 3 Diagram for the effect of different Ganoderic acid components on AFB1-induced DNA damage in HepG2 cells; A: Effect of pretreatment with Ganoderic acids A, B, C, D, F, J on the content of γ-H2AX in HepG2 cells induced by AFB1, B: Effect of pre-incubation with Ganoderic acid B for 0, 6, 12, 24 h on the content of γ-H2AX in HepG2 cells induced by AFB1;

[0014] Figure 4Figure for immunofluorescence detection of the number of γ-H2AX foci; A: Immunofluorescence staining analysis of γ-H2AX foci in the cell nuclei of different treatment groups, B: Quantitative analysis of γ-H2AX foci in the cell nuclei of different treatment groups (*p<0.05; **p<0.01; ***p<0.001). Detailed implementation mode

[0015] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art in the technical field concerned understands the embodiments of the content of the present invention, the technology taught by the content of the present invention can be changed and modified, which does not depart from the spirit and scope of the content of the present invention.

[0016] The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but are not used to limit the present invention.

[0017] Embodiment

[0018] 1. Cell resuscitation and culture

[0019] Cell resuscitation:

[0020] The cryopreservation tube is taken out of the liquid nitrogen tank and placed in a water bath at 37°C. The cryopreservation tube is gently shaken to quickly melt it. It is taken out, wiped clean and transferred to the ultra-clean workbench for operation. The cell (liver cancer cell line HepG2) suspension in the tube is transferred to a 1.5 mL centrifuge tube with a pipette gun, centrifuged at 1250 rpm for 5 minutes. After removing the supernatant, the cells are resuspended by pipetting with 1 mL of complete medium, transferred to a 6 cm culture dish containing 2 mL of complete medium, and mixed by the cross method. It is cultured in an incubator at 37°C and 5% CO2 for 24 hours. The next day, the original medium is discarded, and the cells are washed once with 1 mL of PBS to wash away the necrotic cells and debris. Then 3 mL of complete medium is added and the culture is continued. When the confluence reaches about 80%, subculture and amplification are carried out.

[0021] Cell culture:

[0022] The following culture medium formula is used for the human liver cancer cell line HepG2: 10% FBS, 1% GlutaMax and 1% double antibody (penicillin-streptomycin) are added by volume percentage, and finally the high-glucose medium DMEM is used to make up the volume.

[0023] Cell passage:

[0024] Place the cells with a fusion degree of 75-85% on the ultra-clean workbench. Carefully aspirate the supernatant with a pipette, add 2 mL of PBS to wash away the residual culture medium, and repeat once. Then add 1 mL of diluted trypsin (0.25% trypsin:PBS = 1:1). Observe the cell digestion status under the microscope. After sufficient digestion, discard the trypsin with a pipette gun and add 1 mL of culture medium to pipette the cells into a suspension state. According to the set passage ratio of 1:2, take out 500 μL of cell suspension and add it to a 6-cm culture dish containing 2.5 mL of complete medium. Shake it evenly by the cross method and transfer it to an incubator (37 °C, CO2 concentration 5%) for culture. When the number of adherent cells reaches about 80%, passage and amplify again.

[0025] 2. Cell viability detection

[0026] Take 4×10 3 cells and inoculate them into a 96-well plate at 100 μL / well, and place them in an incubator for 12-24 h. Dilute the AFB1 with a mother liquor concentration of 80 mmol / L to working solutions with final concentrations of 0, 10, 20, 40, 60, 80, and 100 μmol / L with complete medium. Each group has three replicate wells, add 100 μL of medium containing AFB1 to each well, and place it in the incubator for further culture for 24 h. Perform cell viability determination by the CCK-8 method. Prepare the CCK-8 detection solution and complete medium in a ratio of 1:9, then pipette and mix well. Aspirate the complete medium in the wells, wash twice with PBS, add 100 μL of the prepared CCK-8 detection solution to the wells, try to avoid generating bubbles, wrap the 96-well plate with tin foil for light shielding treatment and then place it in the incubator for 0.5-1 h. After the CCK-8 detection reagent changes color during culture, measure the cell absorbance (OD450) at 450 nm with a multifunctional microplate reader (Tecan).

[0027] 3. Western blot analysis

[0028] Seed 1.8×10 5Cells per well were pretreated with a medium containing 10 μmol / L ganoderic acids A, B, C, D, F, and J for 12 h, and then 60 μmol / L AFB1 was added and incubated for 24 h for immunoblotting experiments. The medium was discarded, and the cells were washed twice with PBS. RIPA lysis buffer was added on ice and the cells were lysed for 10 min. The lysate and 5×SDS loading buffer were mixed at a volume ratio of 4:1 and heated in a metal bath for 10 min. SDS-PAGE electrophoresis was performed. 10% separating gel and 10% stacking gel were prepared according to the formula, and samples were loaded. The stacking gel was run at 80 V first, and then the separating gel was run at 150 V. For the transfer membrane electrophoresis, the transfer parameters were set as follows: γ-H2AX: 270 mA, 25 min; β-actin: 300 mA, 1 h, and the transfer was performed in an ice bath. The NC membrane was taken out and placed in 5% skim milk (phosphorylated proteins were blocked with 5% BSA), at 37 °C for 1 h. The antibodies were diluted with the primary antibody diluent in proportion (β-actin at 1:1000, γ-H2AX at 1:1000), incubated overnight at 4 °C, and washed 3 times with 1×TBST, 5 min each time. The secondary antibody (1:3000) was added according to the species of the primary antibody, at 37 °C for 1 h, and washed 3 times with 1×TBST, 5 min each time. Exposure: Exposed and photographed with a chemiluminescence imager.

[0029] 4. Immunofluorescence staining

[0030] 3×10 4 Cells per dish were seeded in a laser confocal dish. On the second day, 10 μmol / L ganoderic acids B and C were added and pretreated for 12 h, and then 60 μmol / L aflatoxin was added and incubated for 24 h for immunofluorescence staining. The medium was discarded, and the cells were washed twice with PBS. Fixed with 4% PFA at room temperature for 20 min, and washed 3 times with PBS, 5 min each time. Permeabilized with 0.3% Triton X-100 at 37 °C for 20 min, and washed 3 times with PBS, 5 min each time. Antigen retrieval solution was used at room temperature for 5 min, and washed 3 times with PBS, 5 min each time. Blocked with 10% DSR at 37 °C for 1 h. The primary antibody (γ-H2AX: 1:800) was incubated overnight at 4 °C, and washed 3 times with PBS, 5 min each time. The fluorescent secondary antibody was diluted with 1% BSA at a ratio of 1:1000, protected from light, incubated at 37 °C for 1 h and at room temperature for 2 h, and washed 3 times with PBS, 5 min each time. The cell nuclei were stained with DAPI at room temperature for 20 min, and washed 3 times with PBS, 5 min each time. Photographed with a fluorescence microscope; the number of γ-H2AX foci was counted using Image J software.

[0031] 5. Safety evaluation of ganoderic acid drugs

[0032] 4×10 3Cells were seeded at 100 μL per well into a 96-well plate and cultured in an incubator for 12 - 24 h. The cells were then treated with Ganoderic acids A, B, C, D, F, and J at different concentrations (0, 4, 8, 16, 32, 64 μmol / L) for 48 h, and cell viability was determined by the CCK-8 method. The detection method was the same as in 2.

[0033] 6. Screening of Ganoderic acids with protective effects against AFB1 damage

[0034] A quantity of 4×10 3 Cells were seeded at 100 μL per well into a 96-well plate and cultured in an incubator for 12 - 24 h. The mother liquor of Ganoderic acids A, B, C, D, F, and J with a concentration of 10 mmol / L was diluted with complete medium to a working solution with a final concentration of 10 μmol / L. In this study, a pretreatment method with Ganoderic acids was used to explore the preventive and protective effects of Ganoderic acids against AFB1 damage. If the cells were first treated with AFB1 and then with Ganoderic acids, the repair effect of Ganoderic acids on AFB1 damage was mainly detected. The cells were pretreated with medium containing Ganoderic acids for 12 h. After discarding the old medium, 100 μL of medium containing 60 μmol / L AFB1 (containing the same concentration of Ganoderic acid components) was added, and the cells were further cultured in an incubator for 24 h. Cell viability was determined by the CCK-8 method. The detection method was the same as in 2.

[0035] 7. Statistical analysis

[0036] All data results were statistically analyzed using GraphPad Prism 6 software. Unpaired t-tests were used to compare differences between groups, and p < 0.05 was considered statistically significant. Note: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Detailed implementation methods

[0038] 1. The laminar flow hood and the aseptic room were sterilized by ultraviolet irradiation for 40 min. Then, the ultraviolet equipment was turned off, and the exhaust system was turned on. Enter the aseptic room after 5 min.

[0039] 2. Turn on the water bath and adjust the water temperature to 37 °C to preheat the medium.

[0040] 3. Take the liver cancer cells cryopreserved in the laboratory from the liquid nitrogen tank, quickly thaw them by shaking in a 37 °C water bath, centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend the cells with 1 mL of proliferation medium, inoculate them into a 6 cm cell culture dish, add 3 mL of proliferation medium, and culture them in a 37 °C incubator.

[0041] 4. Observe the resuscitated and cultured cells daily. When the cells reach 80% confluence, take out the 6-cm cell culture dish, add 2 mL of PBS and wash twice. After removing the residual PBS, add a mixed solution of 200 μL of trypsin and 1 mL of PBS, place it in a 37°C incubator for digestion for 1 minute, add 1 mL of proliferation medium, gently pipette to detach the cells from the wall, collect the cell suspension into a 15-mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, then remove the supernatant, and inoculate and passage at a ratio of 1:3, and expand the culture in a 37°C 5% CO2 incubator.

[0042] 5. Culture the resuscitated cells to the third generation, perform cell counting after washing, digestion, and centrifugation according to the passage method, passage to a 96-well plate at a quantity of 4000 cells per well, add 100 μL of medium to each well, and continue to culture in a 37°C 5% CO2 incubator.

[0043] 6. The next day, add different concentrations of AFB1 (0, 10, 20, 40, 60, 80, 100 μmol / L) according to the experimental design to treat human hepatocellular carcinoma HepG2 cells for 24 hours, or add different concentrations of ganoderic acid (0, 4, 8, 16, 32, 64 μmol / L) to treat the cells for 48 hours, or pretreat the cells with 0, 5, 10, 20, 40 μmol / L ganoderic acid B for 12 hours and then treat the cells with AFB1 for 24 hours; then detect cell viability by the CCK8 method.

[0044] 7. Prepare cells separately, seed 1.8×10 5 cells per well in a six-well plate, pretreat the cells with medium containing 10 μmol / L ganoderic acid A, B, C, D, F, J for 12 hours, and then add 60 μmol / L AFB1 to treat for 24 hours for immunoblotting experiments.

[0045] 8. Prepare cells separately, inoculate 3×10 4 cells per dish in a confocal dish, add 10 μmol / L ganoderic acid B and C the next day to pretreat for 12 hours, and then add 60 μmol / L aflatoxin to treat for 24 hours for immunofluorescence staining.

[0046] 9. According to the above experimental design, each experimental design is repeated 3 times.

[0047] Experimental results:

[0048] 1. Construction of AFB1 damage model

[0049] Treat human hepatocellular carcinoma HepG2 cells with different concentrations of AFB1 (0, 10, 20, 40, 60, 80, 100 μmol / L) for 24 hours, and detect cell viability by the CCK8 method, as Figure 1As shown in , the cell viability gradually decreased with the increase of AFB1 treatment concentration; human liver cancer HepG2 cells were treated with different concentrations of AFB1 (0, 10, 20, 40, 60, 80 μmol / L) for 24 h, and the protein level of DNA damage marker γ-H2AX was detected. As Figure 1 shown in B: with the increase of AFB1 treatment concentration, the protein level of γ-H2AX gradually increased, indicating that the DNA damage caused by AFB1 was concentration-dependent; then, human liver cancer HepG2 cells were treated with 60 μmol / L AFB1 for different time gradients (0, 6, 12, 24 h). As Figure 1 shown in C, the number of γ-H2AX foci gradually increased with the increase of treatment duration, indicating that the DNA damage caused by AFB1 was time-dependent. The above results showed that: treating human liver cancer HepG2 cells with 60 μmol / L AFB1 for 24 h could significantly reduce cell viability and mainly caused DNA damage to cells. Therefore, this concentration was used for subsequent experiments.

[0050] 2. Screening of active components of Ganoderma lucidum

[0051] To screen for Ganoderma lucidum acid components with protective effects against AFB1 damage, we first screened the drug safety concentrations of Ganoderma lucidum acids A, B, C, D, F, and J. Cells were treated with different concentrations of Ganoderma lucidum acid (0, 4, 8, 16, 32, 64 μmol / L) for 48 h, and cell viability was detected. As Figure 2 shown in A, the safe concentration range of Ganoderma lucidum acid was 0 - 16 μmol / L. To detect the preventive and protective effects of Ganoderma lucidum acid on AFB1 damage, this study used the method of Ganoderma lucidum acid pretreatment to explore. Human liver cancer HepG2 cells were pretreated with 10 μmol / L concentrations of Ganoderma lucidum acids A, B, C, D, F, and J for 12 h, and then treated with AFB1 for 24 h. Cell viability was detected by the CCK8 method. As Figure 2 shown in B, compared with the group treated with AFB1 alone, HepG2 cells pretreated with Ganoderma lucidum acid B could restore cell viability to a certain extent, and there was a significant difference (***p < 0.001). To further determine the optimal concentration of Ganoderma lucidum acid B, cells were pretreated with 0, 5, 10, 20, 40 μmol / L Ganoderma lucidum acid B for 12 h, and then treated with AFB1 for 24 h. As Figure 2 shown in C, 10 μmol / L Ganoderma lucidum acid B had the best protective effect against AFB1 damage. The above results suggested that low-concentration Ganoderma lucidum acid B had no obvious toxic and side effects on HepG2 cells and could resist part of the damage caused by AFB1 to cells.

[0052] 3. Mechanism analysis of Ganoderma lucidum acid resisting AFB1-induced cell damage

[0053] To further investigate the mechanism of ganoderic acid resistance to AFB1-induced cell damage, human hepatocellular carcinoma HepG2 cells were pretreated with different ganoderic acids A, B, C, D, F, and J, and the DNA damage marker γ-H2AX was detected by Western blotting, as Figure 3 shown in Figure A, pretreatment with ganoderic acid B could reduce the protein level of γ-H2AX; further, HepG2 cells were pretreated with ganoderic acid B for 0, 6, 12, and 24 h respectively, as Figure 3 shown in Figure B, the protein level of γ-H2AX in the experimental group was significantly decreased after 12 h of pretreatment with ganoderic acid B. The results suggest that ganoderic acid B has a protective effect on AFB1-induced DNA damage in cells.

[0054] 4 Ganoderic acid B treatment reduces AFB1-induced DNA damage in cells

[0055] To further verify the above results, immunofluorescence staining was used. After pretreatment of HepG2 cells with ganoderic acids B and C for 12 h, AFB1 was added and the cells were treated for 24 h, and then the number of γ-H2AX foci in the cell nucleus was detected by immunofluorescence staining, as Figure 4 shown in Figure A. Compared with the group treated with AFB1 alone, the number of γ-H2AX foci in the group pretreated with ganoderic acid B was significantly decreased (***p < 0.001). Therefore, the present invention provides a new method for the protective mechanism of ganoderic acid against AFB1-induced DNA damage in cells. Specifically, 10 μmol / L ganoderic acid B has the best protective effect on AFB1-induced hepatocyte damage. Using the above method in this example, the same experiment was carried out on HL7702 cells, and it was found that ganoderic acid B also has a protective effect on AFB1-induced damage in HL7702 cells, and 10 μmol / L ganoderic acid B has the best effect.

Claims

1. Application of Ganoderic acid B in preparing a drug for treating hepatocyte damage caused by aflatoxin.

2. The application according to claim 1, wherein The aflatoxin described above is aflatoxin B1.

3. The application according to claim 1, wherein The hepatocyte damage described above is DNA damage.

4. The application according to any one of claims 1 to 3, characterized in that, The molar concentration of Ganoderic acid B described above is 5 - 40 μmol / L.

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