Application of ganoderenic acid A in promoting anti-tumor killing function of T lymphocytes

Abstract

The invention discloses application of ganoderenic acid A in enhancing the killing function of T lymphocytes. The ganoderenic acid A is lanolin alkyne alkyl type triterpenoid, and it is proved that the ganoderenic acid A can promote T lymphocytes to release granzyme, perforin and interferon within the concentration range of 5-10 [mu] M, and then the killing function of the T lymphocytes is promoted. The early foundation is laid for the ganoderenic acid A to become an immunoregulation small molecule medicine in the future, and it is indicated that the ganoderenic acid A is expected to become a recognized XinJin immune activator.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of ganoderenic acid A in promoting the anti-tumor killing function of T lymphocytes. Background Art

[0002] Ovarian cancer (OC) is a disease in which ovarian cells undergo pathological changes and form malignant tumors. Since the ovaries are deep in the female abdominal cavity, its symptoms are usually not obvious and it is difficult to detect in the early stage. Often, it is already in the advanced stage when diagnosed. Currently, the treatment methods for ovarian cancer mainly include traditional surgical treatment, chemotherapy, and targeted therapy, etc. According to the latest statistical data, the incidence of ovarian cancer is increasing year by year among female reproductive system tumors, and the mortality rate ranks first among female genital tract malignancies.

[0003] In recent years, the newly emerging immunotherapy has made significant progress in the treatment of ovarian cancer, and immunomodulators have gradually become well-known to humans. An immunomodulator is a preparation that regulates the body's immune system balance and thus regulates the body's immunity. It mainly includes immunostimulants, immunosuppressants, and bi-directional immunomodulators. However, there is little research on immunomodulators at present. In addition, as an immunosuppressive tumor, seeking or developing immunomodulators for the treatment of ovarian cancer has become a current research hotspot.

[0004] Ganoderma lucidum, as a traditional Chinese medicine, has been proven to have the effects of promoting innate and adaptive immunity and anti-tumor, and the main active ingredients playing the role are usually ganoderma polysaccharides or ganoderma triterpenoids. Nowadays, there are numerous reports on the immunomodulation of ganoderma polysaccharides, while for ganoderma triterpenoids, only their direct anti-tumor activity has been proven, and there are almost no reports related to immunity. Therefore, by studying the effects of ganoderma triterpenoids, specifically ganoderenic acid A (GAA), on the anti-tumor killing function of T cells among immune cells, it lays a solid foundation for the future development of small molecule immunomodulators of ganoderma triterpenoids.

[0005] Ganoderenic acid A (GAA) is a lanostane-type triterpenoid isolated from Ganoderma lucidum. It has been reported that ganoderenic acid A has an effective liver protection effect on CCl4-induced liver injury. However, there is no literature and report on the application of ganoderenic acid A in immunity. Summary of the Invention

[0006] The present invention aims to provide the application of GAA in promoting T lymphocytes to secrete granzyme, perforin, and interferon, and prove that GAA is an immune activator, which can improve the anti-tumor killing function of T lymphocytes by promoting T lymphocytes to release granzyme B, perforin, and interferon γ (IFN-γ).

[0007] To achieve the above object, the present invention adopts the following technical solutions: Use of ganoderic acid A in enhancing the killing function of T lymphocytes, the structural formula of the ganoderic acid A is: .

[0008] Furthermore, the ganoderic acid A enhances the killing function of T lymphocytes by promoting the release of T lymphocyte granzyme, perforin and interferon.

[0009] Furthermore, the concentration of ganoderic acid A is 5-10 μM.

[0010] Use of ganoderic acid A in promoting apoptosis of tumor cells, the structural formula of the ganoderic acid A is: .

[0011] Furthermore, the tumor cells are A2780 cells.

[0012] Furthermore, ganoderic acid A indirectly kills A2780 cells by acting on tumor-specific T cells.

[0013] Furthermore, the preparation steps of tumor-specific T cells are: (1) Culturing DC cells in a medium containing 25 ng / mL IFN-γ for 24 h; (2) Co-culturing the DC cells obtained in step (1) with 50 μg / mL A2780 tumor antigen for 24 h to obtain antigen-loaded DC cells; (3) Co-culturing the DC cells obtained in step (2) with T cells at a cell number ratio of 1:5 in complete T cell medium for 24 h to obtain tumor-specific T cells.

[0014] Finally, the present invention also provides the use of ganoderic acid A in the preparation of an immune activator.

[0015] The beneficial effects of the present invention are: The results of the present invention confirm that low concentration of GAA can promote the secretion of granzyme, perforin and interferon by T lymphocytes, enhance the killing ability of T lymphocytes, and thus improve the immunity of the body. GAA can promote the effect of anti-tumor immunotherapy. Description of the Drawings

[0016] Figure 1 : Effects of GAA on the secretion of Granzyme B and Perforin by T lymphocytes; where n = 3, *** p<0.001, **** p<0.0001 vs. Control.

[0017] Figure 2 : Effect of GAA on IFN-γ secretion by T lymphocytes; where n = 3, **** p<0.0001 vs. Control.

[0018] Figure 3 : Effect of tumor-specific T lymphocytes on apoptosis of A2780 cells under the action of GAA; where n = 3, **** p<0.0001 vs. Control. Detailed implementation manners

[0019] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0020] The experimental materials involved in the following examples are as follows: Ganoderenic acid A in the following examples was purchased from Wuhan Tianzhi Biotechnology Co., Ltd., product number CFN92236, batch number: CFS202302, purity ≥98%; weighed Ganoderenic acid A was dissolved in DMSO to prepare a mother liquor of Ganoderenic acid A with a concentration of 100 mM, and diluted to the corresponding concentration with complete T lymphocyte medium for the following implementations.

[0021] The CD3 antibody was purchased from Takara, catalog number T210; Ficoll Paque PREMIUM was purchased from Cytiva, catalog number 17544202; Lonza X-VIVO 15 medium was purchased from Lonza, catalog number 04-418Q; fetal bovine serum (FBS) was purchased from Cegrogen, catalog number A0500-3011; IL-2 was purchased from Sino Biological, catalog number GMP-11848-HNAE; IFN-γ was purchased from Sino Biological, catalog number GMP-11725-HNAE; IL-4 was purchased from Sino Biological, catalog number GMP-11846-HNAE-L; GM-CSF was purchased from Sino Biological, catalog number GMP-10015-HNAH; trypsin was purchased from HyClone, catalog number SH30042.02; human ovarian cancer cell line A2780 was purchased from KeyGEN BioTECH, catalog number KGG3271-1; PBS buffer was purchased from ABK Bio, catalog number ABK0001C; 4% paraformaldehyde was purchased from Biosharp, catalog number BL539A; permeabilizer was purchased from BD, catalog number 554714; CD3-PC5, CD4-PC7 and CD8-ECD antibodies were all purchased from Beckman, catalog numbers were A07749, 737660, 6604728 respectively; IFN-γ-FITC, GranzymeB-FITC, Perforin-BV421 antibodies were all purchased from BD, catalog numbers were 340449, 560211, 563393 respectively; cell apoptosis kit was purchased from Beyotime, catalog number C1062.

[0022] Example 1 Extraction and culture of T lymphocytes (1) Preparation of CD3 antibody-coated plate: Dilute the CD3 monoclonal antibody with sterile PBS buffer (pH 7.4) to a final concentration of 10 µg / mL. Add 1 mL of the CD3 monoclonal antibody dilution to each well of a 6-well plate. Wrap the joints of the 6-well plate with sealing film and coat overnight in a 4°C refrigerator.

[0023] (2)Extraction of T lymphocytes: On the second day, 4 mL of peripheral blood was drawn from healthy individuals, centrifuged at 2000 rpm for 10 min, the upper serum was discarded, and the lower blood cells were collected. Then, an equal volume of sterile PBS buffer pre-cooled at 4°C was added and mixed evenly for dilution. 4 mL of Ficoll Paque PREMIUM was added to a new 15 mL centrifuge tube, and the diluted blood cells were slowly added along the inclined wall of the 15 mL centrifuge tube, so that the diluted blood cells were evenly spread on the lymphocyte separation solution. The acceleration of the centrifuge was adjusted to 8 and the deceleration to 0, and centrifuged at 2000 rpm for 20 min. After centrifugation, obvious stratification occurred, divided into four layers from top to bottom: plasma layer (containing platelets), milky white ring layer (i.e., PBMC layer), lymphocyte separation solution layer, and red blood cell layer. The PBMC layer was slowly aspirated with a Pasteur pipette in a circular motion, taking care not to pierce the interface between the two layers. The PBMC was transferred to another 15 mL centrifuge tube containing 10 mL of sterile PBS buffer, centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the cell pellet was collected. Then, it was washed twice with 5 mL of sterile PBS buffer to obtain T lymphocytes.

[0024] (3)An appropriate amount of complete medium for T lymphocytes (Lonza X-VIVO 15 medium + fetal bovine serum with a final concentration of 10% + IL-2 with a final concentration of 25 μg / mL + IFN-γ with a final concentration of 50 μg / mL) was used to resuspend the T lymphocytes to obtain a T lymphocyte suspension. 10 μL of the T lymphocyte suspension was taken, diluted 1:1 with trypan blue staining solution, and counted using a cell counter. The density of T lymphocytes was adjusted to 1×10 6 cells / mL; the CD3 antibody dilution solution in the 6-well plate was removed, and 2 mL of the T lymphocyte suspension was added to each well. The STEMCELL CD3 / CD28 antibody solution was added to each well of the 6-well plate at 25 μL / mL, gently mixed, and placed in an incubator at 37°C and 5% CO2 for culture. During the culture, the cell status was continuously observed and fresh complete medium for T lymphocytes was added in a timely manner. After culturing for about 4 - 5 days, depending on the growth of T lymphocytes, it was transferred to a T75 culture flask for further expansion culture. After 7 days, the T lymphocytes were mature for the following experiments.

[0025] Example 2 Effect of GAA on cytokine secretion by T lymphocytes (1)After obtaining T lymphocytes by the extraction method of Example 1, they were counted using a cell counter, and the cell density was adjusted to 5×10 5Cells were inoculated at a density of

[0026] 2×10⁶ cells / mL into 12-well plates, 2 mL per well. The T lymphocytes were divided into a Control group, a DMSO group, a 5 μM GAA group, and a 10 μM GAA group. In the Control group, cells were induced for 48 h without any other treatment. In the DMSO group, an equal volume of DMSO solution as in the experimental groups was added to the T lymphocytes and induced for 48 h. In the 5 μM GAA group, GAA with a final concentration of 5 μM was added to the T lymphocytes and induced for 48 h. In the 10 μM GAA group, GAA with a final concentration of 10 μM was added to the T lymphocytes and induced for 48 h. All the above cells were cultured in a constant temperature incubator at 37 °C and 5% CO₂.

[0027] (2)When there were 4 - 6 h left until the end of the induction culture, stimulants with a final concentration of 10 ng / mL PMA and 1.41 μM Ionomycin, as well as a blocker with a final concentration of 2.5 μM Monensin, were added to all wells to promote the retention of cytokines in the cells, and the culture continued until the end. Cells were collected into EP tubes according to the groups, centrifuged at 2000 rpm for 5 min to discard the supernatant, and the cell pellet was collected. The cell pellet was washed 2 times with PBS (containing 3% FBS). A CD3 - PC5, CD4 - PC7, CD8 - ECD antibody mixture was prepared by adding 1.5 μL of each antibody and 50 μL of PBS (containing 3% FBS) to each sample tube. 50 μL of the above CD3 - CD4 - CD8 antibody mixture was added to each sample tube, mixed well, incubated in the dark at room temperature for 30 min, then centrifuged at 2000 rpm for 5 min to discard the antibody mixture, and washed 2 times with PBS (containing 3% FBS).

[0028] (4)Prepare IFN-γ-FITC, Granzyme B-FITC, and Perforin-BV 421 antibody diluents respectively. Taking IFN-γ as an example: Prepare the IFN-γ antibody diluent by mixing 50 µL of PBS (containing 3% FBS) and 2 µL of IFN-γ-FITC antibody. Add 50 µL of the above IFN-γ antibody diluent to each sample tube, mix well, incubate in the dark at room temperature for 30 min, then centrifuge at 2000 rpm for 5 min to discard the antibody diluent, and wash twice with PBS (containing 3% FBS). Finally, resuspend the cells with 300 µL of PBS (containing 3% FBS), and then perform flow cytometry analysis.

[0029] Example 3 Effect of GAA on apoptosis of A2780 cells after acting on tumor-specific T lymphocytes (1)Preparation of DC cells: After obtaining the PBMC precipitate as in step (2) of Example 1, resuspend the cells with an appropriate amount of DC cell complete medium (X-VIVO 15 serum-free cell medium + IL-4 at a final concentration of 75 ng / mL + GM-CSF at a final concentration of 60 ng / mL), adjust the cell density to 5×10 5 cells / mL, plate them in a 6-well plate, and culture them in a 37°C, 5% CO2 constant temperature incubator.

[0030] (2)Preparation of A2780 tumor antigen: Digest the A2780 cells in the logarithmic growth phase with trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash 1-2 times with sterile PBS buffer to remove the residual trypsin digestion solution; resuspend the cells with an appropriate amount of sterile PBS buffer, place them in a -80°C ultra-low temperature refrigerator for an appropriate time, then melt them in a 37°C water bath, repeat this 4-5 times, centrifuge at 2000 rpm for 5 min, and transfer the supernatant to another clean and sterile centrifuge tube to obtain the A2780 tumor antigen. Take 1 μL of the obtained antigen and detect the protein concentration by Nanodrop.

[0031] (3)Preparation of tumor-specific T lymphocytes: Add IFN-γ at a final concentration of 25 ng / mL to the DC cells cultured for 5 days, and continue to culture for one day; on the second day, co-culture the above DC cells with A2780 tumor antigen at a final concentration of 50 μg / mL, continue to culture for one day, and then harvest the DC cells; after obtaining T cells as in Example 1 and culturing them in vitro until mature, co-culture them in T cell complete medium for 1 day at a ratio of DC cell number: T cell number = 1:5 to obtain tumor-specific T cells.

[0032] (4)After digesting and terminating the A2780 cells in the logarithmic growth phase with trypsin, adjust the cell density of the cell suspension to 1×10 5cells / mL. Take a clean 6-well plate, add 1 mL of the prepared cell suspension to each well, shake well in a figure-eight pattern, and culture in an incubator at 37°C with 5% CO2 until the cells adhere. Then discard the old culture medium. Divide the A2780 cells into a Control group, a 10 µM GAA group, a tumor-specific T cell group (T Cell), and a 10 µM GAA + tumor-specific T cell group (T Cell + 10 µM GAA). The Control group was induced for 48 h without any other treatment. The 10 µM GAA group was induced for 48 h by adding GAA with a final concentration of 10 µM to the A2780 cells. The tumor-specific T cell group was induced for 48 h by adding 6 tumor-specific T cells to the A2780 cells. The 10 µM GAA + tumor-specific T cell group was induced for 48 h by adding GAA with a final concentration of 10 µM and 6 tumor-specific T cells to the A2780 cells. The cell culture medium for all the above groups was selected as the complete T cell medium, and the final volume was 2 mL. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0033] (5)After the culture is completed, discard the cell supernatant in each well, wash 1 - 2 times with PBS buffer. Add 200 μL of trypsin to each well and digest for 2 min. After terminating the digestion, centrifuge at 1000 rpm for 5 min with an EP tube to collect the cells, wash 2 times with PBS buffer. Resuspend the cells by adding 195 μL of Annexin V-FITC binding solution in the cell apoptosis kit from Beyotime to each EP tube, then add 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution, mix well, incubate in the dark at 25°C for 15 min. After the incubation is completed, place the EP tube on ice for temporary storage and perform flow cytometry detection within 1 h.

[0034] The release of Granzyme B, Perforin, and IFN-γ by T lymphocytes is a marker of the killing ability of T lymphocytes. As Figure 1 shown, compared with the Control group, after treating CD3 + CD8 + killer T lymphocytes with 5 µM GAA and 10 µM GAA, the release amounts of Granzyme B and Perforin both increased. And after treating CD3 + CD8 + killer T lymphocytes with 10 µM GAA, the release amounts of Granzyme B and Perforin were even more, indicating that 10 µM GAA has a stronger effect in promoting the killing function of T lymphocytes.

[0035] From Figure 2It can be seen that compared with the Control group and the DMSO group, after treating T lymphocytes with low-concentration GAA, the release amounts of IFN-γ in CD3 + CD4 + helper T lymphocytes and CD3 + CD8 + cytotoxic T lymphocytes all increased significantly, proving that low-concentration GAA can promote T lymphocytes to secrete the cytokine IFN-γ; among them, after treating with 10 µM GAA, the release amounts of IFN-γ in CD3 + CD4 + helper T lymphocytes and CD3 + CD8 + cytotoxic T lymphocytes were more, indicating that the effect was more significant after treating with 10 µM GAA, and also indicating that GAA is expected to become a new immune activator.

[0036] After treating tumor-specific T cells with 10 µM GAA, its effect on the apoptosis of A 2780 cells is as Figure 3 shown. Compared with the Control group, only when treating A 2780 cells with 10 µM GAA or tumor-specific T cells, the apoptosis rate of A 2780 cells did not increase significantly. However, after stimulating tumor-specific T cells with 10 µM GAA and then treating A 2780 cells, the apoptosis rate of A 2780 cells increased significantly, indicating that GAA does not directly play a role in killing tumor cells, but indirectly kills A 2780 cells by acting on tumor-specific T cells. At the same time, it also indicates that GAA can enhance the killing function of T cells, promote cellular immune activation, and thus improve the body's immunity.

[0037] In summary, low-concentration GAA has the ability to promote T lymphocytes to release Granzyme B, Perforin, and IFN-γ. Among them, 10 µM GAA has the best effect on promoting T lymphocytes to release Granzyme B, Perforin, and IFN-γ, indicating that 10 µM GAA can better promote the killing function of T lymphocytes. At the same time, the above experimental results all indicate that GAA is expected to become a new immune activator.

[0038] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. The application of ganoderic acid A in enhancing the killing function of T lymphocytes is characterized by: The structural formula of the ganoderic acid A is: 。 2. The use according to claim 1, characterized in that: The ganoderic acid A enhances the killing function of T lymphocytes by promoting the release of T lymphocyte granzymes, perforins and interferons.

3. The use according to claim 1, characterized in that: The concentration of ganoderic acid A is 5-10 μM.

4. The application of ganoderic acid A in promoting apoptosis of tumor cells is characterized by: The structural formula of the ganoderic acid A is: 。 5. The use according to claim 4, characterized in that: The tumor cells are A2780 cells.

6. The use according to claim 4, characterized in that: Ganoderic acid A indirectly kills A2780 cells by acting on tumor-specific T cells.

7. The use according to claim 6, characterized in that: The steps for preparing tumor-specific T cells are: (1) DC cells were cultured in a medium containing 25 ng / mL IFN-γ for 24 h; (2) Co-culturing the DC cells obtained in step (1) with 50 μg / mL A2780 tumor antigen for 24 hours to obtain antigen-loaded DC cells; The DC cells obtained in step (2) were co-cultured with T cells at a ratio of 1:5 in T cell complete culture medium for 24 hours to obtain tumor-specific T cells.

8. Application of ganoderic acid A in the preparation of immune activators.

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