Application of ganoderenic acid A in reversing depletion state of T lymphocytes

Abstract

The invention discloses an application of ganoderenic acid A in reversing a depletion state of T lymphocytes. The ganoderenoic acid A is lanolin alkyne alkyl type triterpenoid, it is proved that the ganoderenoic acid A can reverse the depletion state of T lymphocytes within the concentration range of 5-10 [mu] M, the original killing function of the T lymphocytes can be recovered, and an earlier-stage foundation is laid for the ganoderenoic acid A to become an anti-tumor immunoregulation small molecule medicine in the future; the ganoderenic acid A is expected to become an auxiliary medicine in immunotherapy of tumor patients.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of ganoderic acid A in reversing the exhausted state of T lymphocytes. Background Art

[0002] Ovarian cancer (OC) is a disease in which ovarian cells undergo pathological changes and form malignant tumors. As an immunosuppressive tumor, in ovarian cancer, under the continuous stimulation of antigens, the memory of T lymphocytes cannot develop effectively, and T cells become exhausted, which is called T cell exhaustion. T lymphocyte exhaustion is mainly manifested as the exhaustion of CD8 T lymphocytes, which is characterized by the loss of effector function, the high expression of inhibitory receptors, changes in the epigenetic and transcriptional profiles, and different metabolic patterns. The impact of T cell exhaustion on the immune system is significant. First, it causes the immune system to be unable to effectively eliminate pathogens or tumor cells in the body because the exhausted T cells have lost their original effector function. Second, T cell exhaustion may also affect the formation of immune memory, making it impossible for us to produce an effective immune response when encountering the same antigen again in the future. Therefore, seeking or developing immunomodulators for the treatment of ovarian cancer has become a current research hotspot.

[0003] Immunomodulators play an important role in tumor treatment. They exert therapeutic effects through mechanisms such as regulating the function of the immune system to enhance the anti-tumor immune response, reducing the side effects of radiotherapy and chemotherapy, and synergistically acting with other treatment methods. Currently, the existing immunomodulators are expensive and have limited access methods, while a variety of small molecule compounds derived from plants have many advantages, making them adjuvants for traditional cancer treatment methods. Compared with monoclonal therapies, they also have some advantages, such as wide sources, low toxic side effects, and diverse immunomodulatory activities. This is also one of the reasons why many scientists seek natural products and synthetic compounds with immunomodulatory effects.

[0004] Ganoderma lucidum (Curtis) P. Karst. is a traditional medicinal fungus belonging to the family Polyporaceae, commonly known as "Ganoderma grass" or "immortal herb". In traditional Chinese medicine, Ganoderma lucidum is classified as a "superior medicine" and has the effects of replenishing qi and calming the mind, relieving cough and asthma, and prolonging life. Ganoderma lucidum has been proven to enhance innate and adaptive immune responses and has immunomodulatory effects. In addition, Ganoderma lucidum can scavenge free radicals, reduce oxidative stress, and exert antioxidant effects. Modern research has found that Ganoderma lucidum contains a variety of bioactive components, including polysaccharides, triterpenoids, proteins, sterols, etc., and its pharmacological effects are extensive, among which the main ones are Ganoderma lucidum polysaccharides or Ganoderma lucidum triterpenoids.

[0005] Ganoderenic acid A (GAA) is a lanostane triterpenoid isolated from Ganoderma lucidum. It has been reported that Ganoderenic acid A has effective liver protection against CCl4-induced liver injury. However, there is no literature or report on the application of Ganoderenic acid A in reversing the exhausted state of T lymphocytes. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of GAA in reversing the exhausted state of T lymphocytes. GAA can be used as an immune remodeling agent to reverse the exhausted state of CD8 + T lymphocytes and regulate the body's immunity.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: Application of Ganoderenic acid A in reversing the exhausted state of T lymphocytes, and the structural formula of the Ganoderenic acid A is: .

[0008] Further, the concentration of the Ganoderenic acid A is 5-10 μM.

[0009] Further, the exhausted state of T lymphocytes is obtained by culturing T lymphocytes and magnetic beads at a quantity ratio of 1:1 for 4-5 times. The particle size of the magnetic beads is 4.5 μm.

[0010] Further, Ganoderenic acid A reverses the exhausted state of T lymphocytes by reducing the expression of TIM-3 protein.

[0011] Further, Ganoderenic acid A reverses the exhausted state of T lymphocytes by promoting the release of Granzyme B and IFN-γ.

[0012] The present invention also provides the application of the above Ganoderenic acid A in the preparation of an immune activator.

[0013] The beneficial effects of the present invention are: The results of the present invention reveal that GAA can reverse the exhausted state of T lymphocytes, restore the killing ability of T lymphocytes, and thus improve the body's immunity. GAA can reduce the expression of inhibitory receptors of exhausted T lymphocytes and restore the ability of exhausted T cells to release cytokines, thereby reversing the exhausted state of T lymphocytes, which lays a foundation for its possible future use as an adjuvant drug for anti-tumor immunotherapy. Brief Description of the Drawings

[0014] Figure 1 : Comparison of the expression of inhibitory receptors PD-1 and TIM-3 in normal T cells and exhausted T cells; where n = 3, *** p < 0.001, **** p < 0.0001 vs. Control.

[0015] Figure 2 : Effects of GAA on the expression of inhibitory receptor PD-1 in exhausted T lymphocytes; where n = 3, ** p<0.05, **** p<0.0001 vs. Control.

[0016] Figure 3 : Effects of GAA on the expression of inhibitory receptor TIM-3 in exhausted T lymphocytes; where n = 3, **** p<0.0001 vs. Control.

[0017] Figure 4 : Effects of GAA on the release of IFN-γ from exhausted T lymphocytes; where n = 3, *** p<0.001, **** p<0.0001 vs. Control.

[0018] Figure 5 : Effects of GAA on the release of Granzyme B from exhausted T lymphocytes; where n = 3, *** p<0.001, **** p<0.0001 vs. Control. Specific implementation manners

[0019] In order 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%; Weigh Ganoderenic acid A and dissolve it in DMSO to prepare a mother solution of Ganoderenic acid A with a concentration of 100 mM, and dilute it to the corresponding concentration with complete T lymphocyte medium for the following implementations.

[0021] The CD3 / CD28 activation magnetic beads were purchased from ZHL Bio, product number GMP-TL601; Ficoll Paque PREMIUM was purchased from Cytiva, product number 17544202; Lonza X-VIVO 15 medium was purchased from Lonza, product number 04-418Q; fetal bovine serum (FBS) was purchased from Cegrogen, product number A0500-3011; IL-2 was purchased from Sino Biological, product number GMP-11848-HNAE; IFN-γ was purchased from Sino Biological, product number GMP-11725-HNAE; phorbol 12-myristate 13-acetate (PMA) was purchased from MCE, product number 16561-29-8; 12.5% PAGE gel rapid preparation kit was purchased from Yaenzyme Biotech, product number PG113; PD-1 and TIM-3 antibodies were both purchased from Cell Signaling Technology, product numbers 86163T and 45208T respectively; protease inhibitor (PMSF) was purchased from Beyotime, product number ST507-10ml; lysis buffer (RIPA) was purchased from Yaenzyme Biotech, product number PC101; 4% paraformaldehyde was purchased from Biosharp, product number BL539A; permeabilizer was purchased from BD, product number 554714; CD3-PC5, CD4-PC7 and CD8-ECD antibodies were all purchased from Beckman, product numbers A07749, 737660 and 6604728 respectively; IFN-γ-FITC and GranzymeB-FITC antibodies were both purchased from BD, product numbers 340449 and 560211 respectively.

[0022] Example 1 Extraction and culture of exhausted T lymphocytes (1)Extraction of T lymphocytes: Draw 4 mL of peripheral blood from healthy individuals, centrifuge at 2000 rpm for 10 min, discard the upper serum, collect the blood cells in the lower layer, and then add an equal volume of sterile PBS buffer (pH 7.4) pre-cooled at 4°C to the blood cells and mix well for dilution. Add 4 mL of Ficoll Paque PREMIUM to a new 15 mL centrifuge tube, slowly add the diluted blood cells along the inclined wall of the 15 mL centrifuge tube, so that the diluted blood cells are evenly spread on the lymphocyte separation medium. Adjust the acceleration of the centrifuge to 8 and the deceleration to 0, and centrifuge at 2000 rpm for 20 min. After centrifugation, there is an obvious stratification, which is divided into four layers from top to bottom: plasma layer (containing platelets), milky white ring layer (i.e., PBMC layer), lymphocyte separation medium layer, and red blood cell layer. Slowly aspirate the PBMC layer in a circular motion with a Pasteur pipette, taking care not to pierce the separation surface between the two layers. Transfer the PBMC to another 15 mL centrifuge tube containing 10 mL of sterile PBS buffer, centrifuge at 2000 rpm for 10 min, discard the supernatant, collect the cell pellet, and then wash it twice with 5 mL of sterile PBS buffer to obtain T lymphocytes.

[0023] (2)Resuspend the T lymphocytes with an appropriate amount of complete T lymphocyte medium (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) to obtain a T lymphocyte suspension. Take 10 μL of the T lymphocyte suspension, dilute it 1:1 with trypan blue staining solution, and count it with a cell counter. Adjust the density of T lymphocytes to 1×10 6 cells / mL, and add 2 mL of the T lymphocyte suspension to each well of a 6-well plate; Take magnetic beads (particle size of 4.5 μm) with the same number of T lymphocytes into an EP tube containing 1 mL of PBS buffer, mix well, let it stand on a magnetic stand for 1 min, remove the PBS buffer, and complete the washing step of the magnetic beads. Resuspend the magnetic beads with an appropriate amount of complete T lymphocyte medium, and add the magnetic beads to each well of the 6-well plate according to the ratio of the number of T lymphocytes to the number of magnetic beads of 1:1, mix gently, and place it in a 37°C, 5% CO2 constant temperature incubator for culture. When obvious aggregation of T lymphocytes is observed under the microscope after culturing for about 4 - 5 days, remove the magnetic beads and count, and replace the complete T lymphocyte medium containing new magnetic beads to continue the stimulation. During the subsequent culture period, re-count and stimulate every 2 days, continuously observe the cell state and timely supplement fresh complete T lymphocyte medium. After 4 - 5 rounds of magnetic bead-induced stimulation, collect the exhausted T lymphocytes for the following experiments.

[0024] Example 2 Effect of GAA on the expression of co-stimulatory and co-inhibitory proteins on the surface of exhausted T lymphocytes (1)After obtaining exhausted T lymphocytes by the extraction method of Example 1, they were counted by a cell counter, and the cell density was adjusted to 5×10 5 cells / mL with complete T cell medium. They were inoculated into a 12-well plate, 2 mL per well. The exhausted T lymphocytes were divided into a Control group, a DMSO group, a PMA group, and a 5 µM GAA group. The Control group was induced for 48 h without any other treatment. The DMSO group was induced for 48 h by adding an equal volume of DMSO solution to the exhausted T lymphocytes. The PMA group was induced for 48 h by adding PMA with a final concentration of 5 µM to the exhausted T lymphocytes. The 5 µM GAA group was induced for 48 h by adding GAA with a final concentration of 5 µM to the exhausted T lymphocytes. The 10 µM GAA group was induced for 48 h by adding GAA with a final concentration of 10 µM to the exhausted T lymphocytes. The above cells were all cultured in a 37°C, 5% CO2 constant temperature incubator.

[0025] (2)After the induction culture ended, they were counted by a cell counter. The cells were collected in EP tubes according to the groups, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cell precipitate was collected and washed twice with PBS buffer. The cell lysate was prepared according to the ratio of protease inhibitor (PMSF): lysate (RIPA) = 1:100. For every 1×10 6 cells, 80 µL of cell lysate was added. The corresponding volume of lysate was added to each group of EP tubes on an ice box. After lysing for 30 - 40 min, cell protein samples were obtained. According to the ratio of 5×Loading Buffer: cell protein sample = 1:4, 5×Loading Buffer was added to the cell protein sample and mixed evenly to obtain a mixed solution. The above protein was boiled in a 100°C water bath for at least 5 min to fully denature the protein.

[0026] (3)According to the instructions of the 12.5% PAGE gel rapid preparation kit, the stacking gel and separating gel were prepared. The separating gel and stacking gel were respectively transferred between two cleaned and clamped glass plates. A comb was horizontally inserted into the stacking gel with both hands. After the gel solidified, the glass plates were removed and placed into a vertical electrophoresis tank. 2 μL of prestained protein Marker (purchased from Yaenzyme Biotech, product number: WJ101) was added to the first loading well, and then 10 μL of protein samples were sequentially added to the loading wells. The initial electrophoresis was carried out at a voltage of 80 V. When the experimental samples ran into a uniform straight line, the electrophoresis voltage was increased to 120 V. After electrophoresis ended, the gel was transferred to a transfer tank for membrane transfer (the membrane transfer condition was electrotransfer at 300 mA for 45 min). After membrane transfer ended, 2.5 g of skim milk powder was dissolved in 50 mL of TBST buffer (pH 7 - 7.4) to obtain 5% skim milk. The bands were soaked in the prepared 5% skim milk and incubated on a shaker at room temperature for 1 h.

[0027] (4)Dilute the antibodies (PD-1 and TIM-3) with 5% defatted milk according to the recommended dilution ratio in the antibody instruction manual. Immerse the above-mentioned strips in the diluted antibody solution and incubate overnight at 4°C. After the incubation of the primary antibody is completed, transfer the strips to TBST buffer (pH 7 - 7.4) and wash them on a shaker for 3 times, 15 minutes each time. After the washing is completed, transfer the strips to the diluted corresponding secondary antibody (rabbit secondary antibody / mouse secondary antibody) and incubate them on a shaker at room temperature for 1 h. After the incubation is completed, wash them with TBST buffer (pH 7 - 7.4) on a shaker for 3 times, 15 minutes each time. After the washing is completed, take out the strips in sequence, and evenly drop the prepared ECL developing solution (Developing Solution A: Developing Solution B = 1:1) on the strips under light-proof conditions to fully cover the strips, and let it stay for about 2 - 3 minutes. Then place the strips in the developer and expose the strips under an appropriate exposure time.

[0028] Example 3 Effect of GAA on Cytokine Secretion by Exhausted T Lymphocytes (1)After obtaining exhausted T lymphocytes by the extraction method same as that in Example 1, count them with a cell counter and adjust the cell density to 5×10 5 cells / mL, inoculate them into a 12-well plate, 2 mL per well. Divide the exhausted T lymphocytes into Control group, DMSO group, PMA group, 5 µM GAA group, and 10 µM GAA group; the Control group is induced for 48 h without other treatment, the DMSO group is added with the same volume of DMSO solution as the experimental group to the T lymphocytes and induced for 48 h, the PMA group is added with PMA with a final concentration of 5 µM to the exhausted T lymphocytes and induced for 48 h, the 5 µM GAA group is added with GAA with a final concentration of 5 µM to the exhausted T lymphocytes and induced for 48 h, and the 10 µM GAA group is added with GAA with a final concentration of 10 µM to the exhausted T lymphocytes and induced for 48 h. The above cells are all cultured in a constant temperature incubator at 37°C and 5% CO2.

[0029] (2)When there are 4 - 6 h left until the end of the induction culture, stimulants with final concentrations of 10 ng / mL PMA and 1.41 μM Ionomycin, as well as a blocker with a final concentration of 2.5 μM Monensin, are added to all wells to promote the retention of cytokines in the cells, and the culture is continued until the end; collect the cells in EP tubes according to the groups, centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and wash it 2 times with PBS (containing 3% FBS). Prepare a CD3 - CD4 - CD8 antibody mixture by adding 1.5 µL of CD3 - PC5, 1.5 µL of CD4 - PC7, 1.5 µL of CD8 - ECD antibody and 50 µL of PBS (containing 3% FBS) to each sample tube. Add 50 µL of the above CD3 - CD4 - CD8 antibody mixture to each sample tube, mix well, incubate in the dark at room temperature for 30 min, then centrifuge at 2000 rpm for 5 min, discard the antibody mixture, and wash 2 times with PBS (containing 3% FBS).

[0030] (3)Add 1 mL of 4% paraformaldehyde solution pre - cooled at 4℃ to each EP tube, mix well, and fix in the 4℃ refrigerator for 24 h. The next day, centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and wash the cell pellet 1 - 2 times with PBS (containing 3% FBS). Add 200 µL of permeabilization solution to each of the 4 groups, gently mix well, wrap it with tin foil and incubate in the dark at 4℃ for 20 min. After the incubation is over, centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and wash 2 times with PBS (containing 3% FBS).

[0031] (4)Prepare IFN - γ - FITC and Granzyme B - FITC antibody dilutions respectively. Taking IFN - γ as an example: Prepare an IFN - γ antibody dilution 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 dilution to each sample tube, mix well, incubate in the dark at room temperature for 30 min, then centrifuge at 2000 rpm for 5 min, discard the antibody dilution, and wash 2 times with PBS (containing 3% FBS). Finally, resuspend the cells with 300 µL of PBS (containing 3% FBS), and then analyze by flow cytometry.

[0032] The expression of co-inhibitory molecule proteins on the surface of T lymphocytes depleted by GAA was detected by Western blot to further explore the ability of GAA to reverse exhausted T lymphocytes. PD-1 and TIM-3 are co-inhibitory molecules on the surface of T lymphocytes, which have the function of negatively regulating T lymphocytes. They can down-regulate the immune response of T lymphocytes, inhibit the inflammatory response mediated by T lymphocytes, thereby reducing the killing ability of T lymphocytes against tumors and causing exhaustion or tolerance.

[0033] Compared with normal T cells, exhausted T lymphocytes are characterized by the high expression of inhibitory proteins PD-1 and TIM-3. As Figure 1 shown, compared with the expression of PD-1 and TIM-3 on the surface of normal T cells, the expression of PD-1 and TIM-3 on exhausted T cells was significantly increased, indicating that the induction of exhausted T lymphocytes was successful.

[0034] After treating exhausted T lymphocytes with low-concentration GAA for 48 h, the protein expression of exhausted T lymphocytes was detected by Western blot. As Figure 2 shown, compared with the Control group, the expression level of the co-inhibitory protein PD-1 on the surface of exhausted T lymphocytes treated with low-concentration GAA was significantly down-regulated, and the down-regulation effect of PD-1 expression level was the best after treatment with 5 μM GAA; as Figure 3 shown, the expression of TIM-3 on the surface of exhausted T lymphocytes treated with 5 μM GAA was significantly reduced, indicating that 5 μM GAA can effectively reverse the exhausted state of T lymphocytes; as Figure 4 shown, compared with the Control group, the ability of exhausted T lymphocytes to release IFN-γ was significantly enhanced after treatment with low-concentration GAA, and the ability to release IFN-γ was the best after treatment with 5 μM GAA; indicating that 5 μM GAA can effectively reverse the exhausted state of T lymphocytes and restore the killing ability of CD8 T lymphocytes; as Figure 5 can be seen, after treating exhausted T lymphocytes with 5 μM GAA, the ability of exhausted T lymphocytes to resume secreting Granzyme B was restored, and the release amount was significantly increased compared with the Control group; in summary, 5 μM GAA can effectively reverse the exhausted state of T lymphocytes, restore the killing ability of exhausted T cells, and increase the release amounts of IFN-γ and Granzyme B in exhausted T cells.

[0035] All of the above results indicate that 5 μM GAA can further restore the killing function of T lymphocytes by reversing the exhausted state of T lymphocytes, thereby improving the body's immunity, and at the same time laying a foundation for Ganoderenic acid A to become an adjuvant drug in the immunotherapy of tumor patients.

[0036] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. The application of ganoderic acid A in reversing the exhaustion state 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 concentration of the ganoderic acid A is 5-10 μM.

3. The use according to claim 1, characterized in that: The T lymphocyte exhaustion state was obtained by culturing T lymphocytes and magnetic beads at a quantity ratio of 1:1 for 4 to 5 times, and the particle size of the magnetic beads was 4.5 μm.

4. The use according to claim 1, characterized in that: Ganoderic acid A reverses the T lymphocyte exhaustion state by reducing the expression of TIM-3 protein.

5. The use according to claim 1, characterized in that: Ganoderic acid A reverses the T lymphocyte exhaustion state by promoting the release of Granzyme B and IFN-γ.

6. Application of Ganoderic A in the preparation of immune activators.

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