Application of beta-ionone in preparation of tumor immunomodulator

Abstract

The invention relates to application of beta-ionone in preparation of a tumor immunomodulator, and belongs to the technical field of biological medicines. In order to solve the problems of PD-L1 mediated immune escape and insufficient T cell killing activity in existing gastric cancer immunotherapy, the invention provides application of beta-ionone in preparation of a tumor immunomodulator, the tumor immunomodulator is a tumor immune checkpoint inhibitor, and a tumor immune checkpoint is PD-L1; and the tumor immunomodulator is a tumor immunosensitizer. The invention discovers and verifies that beta-ionone has dual immune regulation and control capabilities of immune checkpoint inhibition and immunotherapy sensitization for the first time, and the beta-ionone as an immune checkpoint inhibitor can significantly down-regulate the expression of PD-L1 and weaken immune escape; as an immunotherapy sensitizer, the compound can effectively activate T cells and optimize a tumor immune microenvironment, can significantly enhance the killing effect of the T cells on gastric cancer cells in in-vivo and in-vitro experiments, and has wide clinical application prospects and conversion values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of β-ionone in the preparation of tumor immunomodulators. Background Technology

[0002] Gastric cancer is the fifth most common cancer and the third leading cause of cancer death worldwide, urgently requiring effective prevention and treatment strategies. Tumor immunotherapy is a novel cancer treatment approach distinct from traditional surgery, chemotherapy, radiotherapy, and targeted therapy. Its core mechanism involves activating the body's own immune system to specifically combat tumor cells, breaking the tumor cells' suppression of the immune system and exerting an immune-killing effect. Immune checkpoints are immunosuppressive molecules expressed on the surface of immune cells, precisely regulating the degree of immune activation and playing a crucial role in preventing autoimmune reactions. Tumor immune checkpoint inhibitors are therapeutic drugs developed targeting immune checkpoints. Their core mechanism of action is to block the binding between immune checkpoint molecules and their ligands, relieving the suppression of T cells, restoring the T cells' ability to recognize and kill tumor cells, and thus achieving the goal of cancer treatment.

[0003] Immunotherapy sensitizers are a class of substances that can increase the body's sensitivity to tumor immunotherapy. Their mechanisms of action are mostly through regulating the tumor immune microenvironment, enhancing the activity of immune cells, and downregulating the expression of molecules related to tumor cell immune escape, thereby improving the efficacy of immunotherapy. They are especially suitable for tumor patients who do not respond well to single immunotherapy.

[0004] In the field of gastric cancer immunotherapy, although immune checkpoint inhibitors, represented by programmed death-ligand-1 (PD-L1) monoclonal antibodies, have shown certain therapeutic potential, there are still many problems to be solved in existing treatment regimens: First, PD-L1 is highly expressed on the surface of gastric cancer cells, which makes it easy for tumor cells to escape the immune system by binding PD-L1 to PD-1 on the surface of T cells, thus weakening the effect of immunotherapy; Second, simply downregulating PD-L1 expression is not enough to solve the inhibition of T cells by the tumor microenvironment, and cannot effectively enhance the specific killing activity of T cells against gastric cancer cells, resulting in a low treatment response rate.

[0005] Given the current situation, finding safe and effective immunotherapeutic sensitizers to improve the response of gastric cancer patients to immunotherapy has become an important direction in current gastric cancer treatment research. Natural phytochemicals, due to their advantages such as low toxicity and diverse biological activities, have become important candidate sources for developing immunotherapeutic sensitizers. Exploring their sensitizing effects and mechanisms in gastric cancer immunotherapy is of great significance for developing novel gastric cancer treatment regimens. Summary of the Invention

[0006] To address the issues of PD-L1-mediated immune escape and insufficient T-cell killing activity in existing gastric cancer immunotherapy, this invention provides the application of β-ionone in the preparation of tumor immunomodulators.

[0007] The technical solution of the present invention:

[0008] The application of β-ionone in the preparation of tumor immunomodulators, wherein the mass percentage of β-ionone in the tumor immunomodulator is 0.05~99.5%.

[0009] Furthermore, the tumor immunomodulator also contains pharmaceutically acceptable excipients.

[0010] Furthermore, the tumor immunomodulator is an injectable formulation.

[0011] Furthermore, the tumor immunomodulator is a tumor immune checkpoint inhibitor, and the tumor immune checkpoint is PD-L1.

[0012] Furthermore, the tumor immunomodulator is a tumor immunosensitizer, and the tumor immunosensitization is mediated by T cells.

[0013] Furthermore, the tumor immunomodulator has at least one of the following uses:

[0014] (1) Downregulate the expression of PD-L1 in tumor cells and weaken tumor immune escape;

[0015] (2) Enhance CD8 + Antitumor activity of cytotoxic T cells;

[0016] (3) Downregulates the activity of the Wnt / β-catenin signaling pathway in tumor cells;

[0017] (4) Activate the immune response of the tumor microenvironment and synergistically enhance the killing effect of T cells.

[0018] Furthermore, the tumor is gastric cancer.

[0019] The beneficial effects of this invention are:

[0020] This invention is the first to discover and demonstrate that β-ionone (BI) possesses dual immunomodulatory capabilities of both immune checkpoint inhibition and immunotherapy sensitization, overcoming the limitations of single-function immunotherapeutic drugs and providing a novel approach to tumor immunotherapy. As an effective immune checkpoint inhibitor, BI can significantly downregulate the expression of the key immune checkpoint protein PD-L1 in gastric cancer cells, directly weakening the immune escape ability of tumor cells. Simultaneously, as an immunotherapy sensitizer, it can effectively activate T cells and enhance the tumor-killing function of CD8+ cells. +The proportion of T cells in total T cells and the IFN-γ secretion capacity were optimized to improve the tumor immune microenvironment and support T cell killing function. In both in vivo and in vitro experiments, BI significantly enhanced the killing effect of T cells on gastric cancer cells, achieving a synergistic effect of "direct tumor suppression + enhanced immunity".

[0021] The dual immunomodulatory efficacy of this invention is achieved through precise inhibition of the transcriptional activity of the Wnt / β-catenin signaling pathway. This not only has a clear mechanism of action and strong targeting, but also effectively addresses the problem of resistance to PD-1 / PD-L1 inhibitors in some tumors caused by abnormal activation of this pathway, enabling previously insensitive tumors to regain a response. Based on this, BI can be used alone to prepare tumor immune checkpoint inhibitors, or as a sensitizer in combination with existing PD-L1 inhibitors to enhance therapeutic efficacy. This provides experimental evidence for the development of a new generation of highly effective, broad-spectrum tumor immunotherapies, and has broad clinical application prospects and translational value. Attached Figure Description

[0022] Figure 1 The image shows a comparison of cell viability of gastric cancer cells and human gastric mucosal cells treated with different concentrations of BI in the CCK-8 assay in Example 1. A represents MKN45 cells, B represents AGS cells, and C represents GES-1 cells.

[0023] Figure 2 The images show a comparison of colony formation in MKN45 and AGS cells treated with different concentrations of BI for 72 hours, as detected in Example 1. A is a comparison of colony photos, B is a statistical chart of the number of MKN45 cell colonies, and C is a statistical chart of the number of AGS cell colonies.

[0024] Figure 3 The following are comparative graphs of cell cycle distribution after 72 hours of treatment with different concentrations of BI as detected by flow cytometry in Example 1: A is a comparative graph of cell cycle distribution, B is a statistical graph of cell cycle distribution of MKN45 cells, and C is a statistical graph of cell cycle distribution of AGS cells.

[0025] Figure 4 The image shows a comparison of the expression of PD-L1, PD-1, pGSK-3β (inactive form of GSK-3β protein), GSK-3β and internal reference protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in MKN45 cells after 72 hours of treatment with different doses of BI in Example 2. A is the Western blot result image, and B is the statistical graph of relative protein expression levels.

[0026] Figure 5The image shows a comparison of the expression of PD-L1, PD-1, pGSK-3β, GSK-3β and GAPDH proteins in MKN45 cells after BI treatment for 24, 48 and 72 h in Example 2, detected by Western blot. A is the Western blot result image and B is the statistical graph of relative protein expression levels.

[0027] Figure 6 This is a comparison of the expression of PD-L1, pGSK-3β, GSK-3β and GAPDH proteins in MKN45 cells from different groups after LiCl pretreatment for 6 h and BI treatment for 72 h, as detected by Western blot in Example 2. A is the Western blot result and B is the statistical graph of relative protein expression levels.

[0028] Figure 7 The image shows a comparison of cell viability after co-culturing MKN45 cells or AGS cells with T cells at different ratios for 72 hours, as determined by the CCK-8 assay in Example 3. A represents MKN45 cells, and B represents AGS cells.

[0029] Figure 8 This is a comparison of cell viability of T cells treated with different concentrations of BI for 72 hours, as determined by the CCK-8 assay in Example 3.

[0030] Figure 9 For example, in Example 3, flow cytometry was used to detect CD8+ in T cells treated with BI for 24 hours. + T cells and CD4 + Comparison of T cell subset proportions, A represents the CD3+ content in T cells of the 0 μmol / L BI treatment group. + / CD8 + Flow cytometry scatter plot of subpopulation distribution, B represents CD3 in T cells of the 100 μmol / L BI treatment group. + / CD8 + Flow cytometry scatter plot of subpopulation distribution, C is CD3 in T cells of the 0 μmol / L BI treatment group. + / CD4 + Flow cytometry scatter plot of the subpopulation, D represents CD3 in T cells of the 100 μmol / L BI treatment group. + / CD4 + Flow cytometry scatter plots of the subpopulations, with E for 0 μmol / L and 100 μmol / L BI treatment groups CD8. + T cells and CD4 + A statistical chart of the proportions of T cell subsets;

[0031] Figure 10 This is a comparison of the levels of cytokines IFN-γ and TNF-α in T cells treated with BI for 24 hours, as detected by flow cytometry in Example 3.

[0032] Figure 11 The images shown in Example 4 are comparisons of cell viability after co-culturing gastric cancer cells and T cells in different treatment groups for 72 hours, as detected by the CCK-8 assay. A represents the activity comparison of MKN45 cells and T cells co-cultured at a ratio of 1:2.5; B represents the activity comparison of MKN45 cells and T cells co-cultured at a ratio of 1:5; C represents the activity comparison of AGS cells and T cells co-cultured at a ratio of 1:2.5; and D represents the activity comparison of AGS cells and T cells co-cultured at a ratio of 1:5.

[0033] Figure 12 The images shown are comparisons of colony formation in different treatment groups after co-culturing gastric cancer cells and T cells for 72 hours, as detected by the colony formation experiment in Example 4. A is a comparison of colony photos, B is a statistical chart of the number of MKN45 cell colonies, and C is a statistical chart of the number of AGS cell colonies.

[0034] Figure 13 This is a comparison of the expression of PD-L1, PD-1 and GAPDH proteins in MKN45 cells of each group after 72 h of BI combined with T cell treatment in Example 5, detected by Western blot. A is the Western blot result and B is the statistical graph of relative protein expression.

[0035] Figure 14 This is a comparison of the expression of β-catenin, pGSK-3β, GSK-3β and GAPDH proteins in MKN45 cells of each group after 72 h of BI combined with T cell treatment in Example 5, detected by Western blot. A is the Western blot result and B is the statistical graph of relative protein expression.

[0036] Figure 15 This is a comparison of the expression of pGSK-3β, GSK-3β, PD-L1 and GAPDH proteins in MKN45 cells after LiCl pretreatment and BI combined with T cell treatment for 72 h, as detected by Western blot. A is the Western blot result, and B is the statistical graph of relative protein expression.

[0037] Figure 16 The graphs show the comparison of body weight, tumor weight, and tumor volume of the two groups of mice in Example 6. A is the curve of body weight change, B is the comparison of final tumor weight, C is the curve of tumor volume growth, and D is the comparison of final tumor volume.

[0038] Figure 17 The images show a comparison of peripheral blood cell analysis results from two groups of mice in Example 6. A shows a comparison of white blood cell counts, and B shows a comparison of lymphocyte counts.

[0039] Figure 18 For the flow cytometry detection of CD8 in peripheral blood of two groups of mice in Example 6 + T cells and CD4 + Comparison of T cell subset proportions, A represents CD3+ T cells in peripheral blood T cells of control mice. + / CD4 + Flow cytometry scatter plot of subpopulation distribution, B represents CD3+ in peripheral blood T cells of control mice. + / CD8 + Flow cytometry scatter plot of subpopulation distribution, where C represents CD3+ in peripheral blood T cells of mice in group BI. + / CD4 + Flow cytometry scatter plot of subpopulation distribution, where D represents CD3 in T cells of group BI. + / CD8 + Flow cytometry scatter plot of subpopulation distribution, where E represents CD8 in the control group and BI group. + T cells and CD4 + A statistical chart of the proportions of T cell subsets;

[0040] Figure 19 This is a comparison of the expression of PCNA, Cyclin D1, CDK4 and the internal reference protein β-actin in the xenograft tissues of two groups of mice detected by Western blot in Example 7. A is the Western blot result and B is the statistical graph of relative protein expression.

[0041] Figure 20 This is a comparison of the expression of β-catenin, pGSK-3β, GSK-3β and β-actin proteins in the xenograft tissues of two groups of mice detected by Western blot in Example 7. A is the Western blot result and B is the statistical graph of relative protein expression.

[0042] Figure 21 The image shows a comparison of the expression of PD-1, PD-L1 and β-actin proteins in two groups of mouse xenograft tissues detected by Western blot in Example 7. A is the Western blot result image, and B is a statistical graph of relative protein expression levels. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0044] In all embodiments of this invention, ImageJ was used to analyze Western blot grayscale values. Data were presented as mean ± standard deviation (SD), suitable for normally distributed variables. Differences between three or more groups were analyzed using one-way / two-way ANOVA, and multiple comparisons were performed using unpaired t-tests. A p-value < 0.05 was considered statistically significant. All experiments were independently repeated three times to ensure reproducibility.

[0045] Example 1

[0046] This embodiment investigated the inhibitory effect of BI on the proliferation of gastric cancer cells and normal cells.

[0047] The CAS number of the BI used in this embodiment is 14901-07-6, C 13 H 20 O, with a molecular weight of 192.2973, has the structural formula 4-(2,6,6-trimethyl-1-cyclohexenyl)-3-buten-2-one, and a purity of GC ≥ 96%. Human gastric cancer cell lines MKN45 and AGS, and normal human gastric mucosal cell line GES-1 were used as test cells. The human gastric adenocarcinoma cell lines MKN45 and AGS, and the normal human gastric mucosal cell line GES-1 were all purchased from Wuhan Pronosei Life Sciences Co., Ltd.

[0048] (I) CCK-8 Experiment

[0049] Logarithmic growth phase MKN45 cells and AGS cells were seeded at 4000 cells / well in 96-well culture plates. After cell attachment, BI was added to each well at final concentrations of 0, 25, 50, 100, 200, 300, or 400 μmol / L. The plates were incubated at 37°C in a 5% CO2 incubator for 24, 48, and 72 h, respectively. Then, 100 μL of CCK-8 reagent was added to each well, and the plates were incubated for another h. The plates were then placed on a shaker for 30 min, and the OD value was measured at 540 nm. The 0-dose BI treatment group was used as the control group, and its cell viability was set as 1.0. The cell viability of the other BI treatment groups was calculated, and a cell viability comparison chart was obtained.

[0050] Simultaneously, GES-1 cells in the logarithmic growth phase were seeded at 4000 cells / well in a 96-well culture plate. After cell attachment, BI was added at final concentrations of 0, 25, 50, 100, 200, 300, or 400 μmol / L, respectively. The plates were incubated at 37°C in a 5% CO2 incubator for 72 h. Then, 100 μL of CCK-8 reagent was added to each well, and the plates were incubated for another h. The plates were then placed on a shaker for 30 min, and the OD value was measured at 540 nm. The 0-dose BI treatment group was used as the control group, and its cell viability was set as 1.0. The cell viability of the other BI treatment groups was calculated, and a cell viability comparison chart was obtained.

[0051] The results are as follows Figure 1 As shown, the activity of MKN45 and AGS cells decreased significantly with prolonged treatment time and increased BI dosage. This indicates that BI inhibited the cell activity of MKN45 and AGS cells in a dose- and time-dependent manner (P<0.01 or P<0.05). EGS-1 cells treated with the same dose of BI for 72 hours showed almost no change in cell activity (P>0.05), indicating that the same dose of BI treatment was not toxic to normal cells.

[0052] (ii) Settlement Formation Experiment

[0053] MKN45 and AGS cells were cultured at 600 cells / well in six-well plates. After cell attachment, BI was added to final concentrations of 0, 50, 100, 200, 300, or 400 μmol / L, respectively. The plates were incubated at 37°C with 5% CO2 for 72 hours, followed by replacement of the culture medium. The culture medium was then changed every 3 days. After two weeks, the culture was terminated, and 1 mL of 4% paraformaldehyde was added to each well. The plates were then incubated at room temperature for 20 minutes. The paraformaldehyde was removed, and each well was washed three times with PBS to remove residual paraformaldehyde. 1 mL of 0.1% crystal violet solution was added to each well, and the plates were incubated at room temperature for 20 minutes. The crystal violet solution was removed, and each well was washed three times with PBS to remove residual crystal violet solution. Cell colony formation was observed and photographed on the white plate.

[0054] The results are as follows Figure 2 As shown, with increasing BI dosage, the number of colonies of MKN45 cells and AGS cells decreased significantly. When the BI concentration was 100~400 μmol / L, it significantly inhibited the colony formation ability of MKN45 cells and AGS cells (P<0.01), further confirming the inhibitory effect of BI on the proliferation of gastric cancer cells.

[0055] (III) Flow cytometry

[0056] MKN45 cells and AGS cells were cultured to a confluence of 70-80% and seeded at 2.0 × 10⁶ cells per well. 4Cells were incubated with BI at final concentrations of 0, 50, 100, 200, 300, or 400 μmol / L for 72 h at 37°C in a 5% CO2 incubator. MKN45 and AGS cells were then digested and washed twice with PBS. After centrifugation and discarding the PBS, cells were fixed overnight at 4°C with 70% ethanol. Subsequently, the ethanol was discarded, and the cells were washed twice with PBS. Each sample was then stained with 500 μL of propidium iodide fluorescent dye for 30 min in the dark. After centrifugation for 5 min, the staining solution was removed, and the cells were resuspended in 500 μL PBS and transferred to flow cytometry tubes. The DNA content of the samples was analyzed using flow cytometry.

[0057] The results are as follows Figure 3 As shown, BI treatment can arrest the cell cycle of MKN45 cells in the G0 / G1 phase, indicated by the first red peak, and arrest the cell cycle of AGS cells in the S phase. This indicates that BI can inhibit the proliferation of gastric cancer cells by arresting the cell cycle.

[0058] Example 2

[0059] This embodiment verifies the correlation between BI's inhibition of gastric cancer cell proliferation and immune escape, and clarifies that BI inhibits PD-L1 expression by increasing GSK-3β protein activity.

[0060] (i) To investigate the regulatory effect of different concentrations of BI on the expression levels of immune checkpoint proteins and GSK-3β pathway-related proteins in MKN45 cells.

[0061] Logarithmic growth phase MKN45 cells were seeded at 4000 cells / well in 96-well culture plates, and BI was added to a final concentration of 0, 100, 150 or 200 μmol / L, respectively. The plates were incubated at 37℃ in a 5% CO2 incubator for 72 h. The expression levels of immune checkpoint proteins PD-L1, PD-1, pGSK-3β (inactive form of GSK-3β protein), GSK-3β and GAPDH protein were detected by Western blot.

[0062] The specific method for Western blot immunoprotein electrophoresis is as follows:

[0063] MKN45 cells were lysed on ice. After lysis, the cells were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was determined using the BCA method. The sample with the determined total protein content was then added to 5× loading buffer and denatured at 95°C for 5 min. Following denaturation, polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the sample was transferred to a nitrocellulose membrane. The membrane was then blocked with 5% skim milk for 30 min, followed by the addition of primary antibody. The NC membrane was incubated overnight at 4°C on a shaker. The next day, it was washed three times with TBST, incubated with fluorescent secondary antibody at room temperature for 2 h, washed three times with TBST, and then mixed with 1:1 ECL developing solution. The membrane was developed using a protein developer, and protein images were taken. Grayscale analysis was performed using ImageJ software.

[0064] The results are as follows Figure 4 As shown, BI significantly downregulated the expression of PD-L1 and pGSK-3β proteins in a dose-dependent manner (P<0.01 or P<0.05), while having no significant effect on the expression of PD-1 protein.

[0065] (ii) To investigate the regulatory effect of different BI treatment times on the expression levels of immune checkpoint proteins and GSK-3β pathway-related proteins in MKN45 cells.

[0066] Logarithmic growth phase MKN45 cells were seeded at 4000 cells / well in 96-well culture plates, and BI was added to a final concentration of 100 μmol / L. The plates were incubated at 37℃ in a 5% CO2 incubator for 0, 24 h, 48 h or 72 h. The expression levels of immune checkpoint proteins PD-L1, PD-1, pGSK-3β, GSK-3β and GAPDH were detected by Western blot.

[0067] The results are as follows Figure 5 As shown, BI significantly downregulated the expression of PD-L1 and pGSK-3β proteins in a time-dependent manner (P<0.01 or P<0.05), while having no significant effect on the expression of PD-1 protein.

[0068] (III) Introduce GSK-3β activity inhibitors to reverse verify the specificity of BI's mechanism of action.

[0069] Log-phase MKN45 cells were seeded at 4000 cells / well in 96-well culture plates and pretreated with 50 μmol / L GSK-3β activity inhibitor LiCl for 6 h. Then, BI was added to a final concentration of 100 μmol / L and the plates were incubated at 37 °C in a 5% CO2 incubator for 72 h. The expression levels of immune checkpoint protein PD-L1, as well as pGSK-3β, GSK-3β and GAPDH proteins, were detected by Western blot.

[0070] The results are as follows Figure 6 As shown, pretreatment with LiCl, a GSK-3β activity inhibitor, reversed the downregulation of PD-L1 and pGSK-3β proteins by BI (P<0.01). This confirms that BI must activate the GSK-3β pathway to downregulate PD-L1. These results indicate that BI can inhibit PD-L1 expression by increasing GSK-3β protein activity, thereby weakening the immune escape ability of tumor cells and providing a new target for enhancing anti-tumor immune responses.

[0071] In summary, this embodiment demonstrates that β-ionone can act as a tumor immune checkpoint inhibitor. Its core function is to target and regulate the GSK-3β pathway to downregulate the key immune checkpoint molecule PD-L1, thereby breaking tumor immune escape, which is consistent with the core definition of a tumor immune checkpoint inhibitor.

[0072] Example 3

[0073] This embodiment investigated the correlation between BI's inhibition of tumor growth and immune response in the tumor microenvironment, and verified the enhancing effect of BI on T cell activity and tumor killing ability in vitro.

[0074] (a) Obtaining highly active human peripheral blood T lymphocytes

[0075] Methods for isolating and culturing human peripheral blood T lymphocytes: 3 mL of peripheral venous blood was collected from healthy volunteers and placed in a heparin-anticoagulated tube. 6 mL of Ficoll lymphocyte separation medium was added to a 15 mL centrifuge tube. The whole blood was diluted with an equal volume of PBS and slowly added along the tube wall to the 15 mL centrifuge tube. The tube was centrifuged at 1000g for 30 min at room temperature, and the white membrane layer was carefully aspirated using a pipette. The cells were washed twice with PBS, centrifuged at 1000g for 10 min each time. The cells were resuspended in RPMI 1640 medium containing 10% FBS. 25 μL of CD3 / CD28 cell activator (purchased from STEMCELL) and interleukin-2 (IL-2) at a final concentration of 10 ng / mL were added per mL. The cells were then transferred to 12-well plates for culture. Half the medium was replaced every 2-3 days to maintain a cell concentration of 1.0 × 10⁶ cells / mL. 6 T cells / mL, collected in the logarithmic growth phase before use.

[0076] (II) Evaluation of the killing effect of T cells on gastric cancer cells

[0077] Logarithmic growth phase MKN45 cells or AGS cells were seeded at 4000 cells / well in 96-well culture plates. MKN45 cells or AGS cells were co-cultured with activated T cells at cell ratios of 1:0, 1:1, 1:2.5, 1:5, and 1:10. After incubation at 37℃ and 5% CO2 for 24, 48, and 72 h, cell viability was detected using the CCK-8 assay. The MKN45 cell or AGS cell / activated T cell treatment group at a cell ratio of 1:0 was used as the control group, and its cell viability was set as 1.0. The cell viability of the other data groups was calculated to obtain a cell viability comparison chart.

[0078] The results are as follows Figure 7 As shown, with the increase in the number of T cells and the extension of co-culture time, the cell viability of MKN45 cells and AGS cells significantly decreased (P<0.01 or P<0.05). This result clarifies that T cells themselves possess the killing activity against gastric cancer cells.

[0079] (III) Assessing the impact of BI on T cell activity

[0080] Logarithmic growth phase T cells were seeded at 4000 cells / well in 96-well culture plates. BI was added at final concentrations of 0, 25, 50, 100, 200, 300, or 400 μmol / L, respectively. After incubation at 37℃ in a 5% CO2 incubator for 24, 48, and 72 h, cell viability was detected using the CCK-8 assay. The 0-dose BI treatment group was used as the control group, and its cell viability was set as 1.0. The cell viability of the other BI treatment groups was calculated to obtain a cell viability comparison chart.

[0081] The results are as follows Figure 8 As shown, BI treatment has almost no effect on T cell activity, indicating that BI is essentially non-toxic or low-toxic to T cells and does not exert its anti-tumor effect through non-specific cell killing. Therefore, it can be used in combination.

[0082] (iv) To evaluate the regulatory effect of BI treatment on the distribution of human peripheral blood T lymphocyte subsets.

[0083] The distribution of human peripheral blood T lymphocyte subsets after BI treatment was detected by flow cytometry. Specifically, T cells treated with 0 μmol / L BI for 24 h were collected as a control group, and T cells treated with 100 μmol / L BI for 24 h were collected. Cells were washed twice with PBS. 5 μL each of human CD3, CD45, CD4, and CD8 fluorescently labeled flow cytometry antibodies were added sequentially, and the cells were incubated at 4°C in the dark for 30 min. Cells were washed twice with PBS and resuspended in 500 μL PBS. Detection was performed using a BD FACSCalibur flow cytometer, and CD3+ was analyzed using FlowJo software. + / CD4 + and CD3+ / CD8 + T cell ratio.

[0084] The results are as follows Figure 9 As shown, after treatment with 100 μmol / LBI, CD8 + T cells in total T cells (CD3) + The proportion of T cells was significantly increased (P<0.05), CD4 + The proportion of T cells decreased relatively. This indicates that BI treatment can specifically enhance CD8 cells with tumor-killing functions. + The proportion of T cells in total T cells is one of the direct pieces of evidence that BI enhances the anti-tumor immune function of T cells.

[0085] (v) Evaluate the regulatory effect of BI treatment on the secretion of functional cytokines by human peripheral blood T lymphocytes.

[0086] Log-phase MKN45 gastric cancer cells were seeded at 4000 cells / well in 96-well plates and cultured until adherent. Activated human peripheral blood T cells were harvested and the cell concentration was adjusted to 4.0 × 10⁻⁶ cells / well. 4 pcs / mL. Two experimental groups were set up:

[0087] Control group: MKN45 cells and T cells were co-cultured at a ratio of 1:10;

[0088] BI treatment group: MKN45 cells and T cells were co-cultured at a ratio of 1:10, and BI was added at a final concentration of 100 μmol / L.

[0089] After incubation at 37℃ and 5% CO2 for 24 hours, the concentrations of IFN-γ and TNF-α secreted by T cells were detected using an IFN-γ and TNF-α specific ELISA kit. The relative contents of the control group were calculated with the cytokine content as 1.

[0090] The results are as follows Figure 10 As shown, BI treatment also promoted the secretion of key cytokines IFN-γ and TNF-α by T cells, which kill tumors and activate immunity (P<0.05), indicating that BI can enhance the tumor-killing ability of T cells.

[0091] BI treatment can increase the activation of T cells and achieve anti-tumor activity by targeting and regulating the function of immune cells and activating the body's own anti-tumor immune response.

[0092] This embodiment illustrates that β-ionone can be used as an immunotherapeutic sensitizer to enhance T cell activity and improve the activity of core effector cells (CD8+) through targeted regulation. +It increases the proportion of T cells, promotes the secretion of key anti-tumor cytokines, strengthens the anti-tumor function of the body's own immune cells, and activates the immune response in the tumor microenvironment, thereby enhancing the effect of immunotherapy, which is consistent with the characteristics of immunotherapy sensitizers.

[0093] Example 4

[0094] This embodiment investigated the inhibitory effect of BI combined with T cells on the proliferation of gastric cancer cells in vitro.

[0095] (I) CCK-8 assay to investigate the synergistic inhibitory effect of BI and T cells on the proliferation of gastric cancer cells.

[0096] Logarithmic growth phase MKN45 cells and AGS cells were seeded at 4000 cells / well in 96-well culture plates. After cell attachment, four experimental groups were set up:

[0097] ① Control group: Only an equal volume of culture medium was added, without cell treatment;

[0098] ② BI group: Only BI was added to a final concentration of 100 μmol / L;

[0099] ③T cell group: T cells were added at a ratio of MKN45 cells to T cells of 1:2.5 or 1:5, respectively;

[0100] ④BI+T cell group: T cells were added at a ratio of MKN45 cells or AGS cells to T cells of 1:2.5 or 1:5, respectively, and BI was added at a final concentration of 100 μmol / L.

[0101] After incubating each group in a 37℃, 5% CO2 incubator for 72 h, 100 μL of CCK-8 reagent was added to each well, and incubation continued for 1 h. The cells were then placed on a shaker for 30 min, and the OD value was measured at 540 nm. The cell viability of the control group was taken as 1.0, and the cell viability of the other treatment groups was calculated to obtain a cell viability comparison chart.

[0102] The results are as follows Figure 11 As shown, compared with the BI group, T cell group and control group, the BI+T cell group had a more significant inhibitory effect on the proliferation of MKN45 or AGS cells (P<0.01 or P<0.05), and the synergistic inhibitory effect of the 1:5 ratio group was better than that of the 1:2.5 ratio group, indicating that BI can dose-dependently enhance the killing activity of T cells against gastric cancer cells, and the two have a synergistic effect.

[0103] (II) Colony formation assay to verify the synergistic inhibitory effect of BI and T cells on the colony formation ability of gastric cancer cells.

[0104] Logarithmic growth phase MKN45 cells and AGS cells were seeded at 600 cells / well in a six-well plate. After cell attachment, four experimental groups were set up:

[0105] ① Control group: Only an equal volume of culture medium was added, without cell treatment;

[0106] ② BI group: Only BI was added to a final concentration of 100 μmol / L;

[0107] ③T cell group: T cells were added at a ratio of 1:5 between MKN45 cells or AGS cells and T cells, respectively;

[0108] ④BI+T cell group: T cells were added at a ratio of 1:5 between MKN45 cells or AGS cells and T cells, and BI was added at a final concentration of 100 μmol / L.

[0109] All groups were incubated at 37℃ in a 5% CO2 incubator for 72 hours, then the culture medium was replaced with fresh medium. The culture medium was then replaced every 3 days thereafter, and the culture was maintained for two weeks before termination. 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 20 min. The cells were washed 3 times with PBS to remove residual fixative. 1 mL of 0.1% crystal violet solution was added, and the cells were stained at room temperature for 20 min. The cells were washed 3 times with PBS again, dried, and then observed and photographed on a white board.

[0110] The results are as follows Figure 12 As shown, the number of colonies formed in the BI+T cell group was significantly less than that in the BI group, T cell group, and control group (P<0.01). This indicates that the synergistic effect of BI and T cells can not only kill existing gastric cancer cells but also inhibit their subsequent clonal proliferation, thereby weakening the survival and spread of tumor cells from the root.

[0111] This example demonstrates that β-ionone and T cells have a significant synergistic anti-tumor effect. Combined with the conclusions of Example 3, it further corroborates that BI enhances T cell activity by regulating immune cell function, and ultimately achieves highly efficient anti-tumor effects in synergy with T cells, thus clarifying its efficacy as an immunotherapy sensitizer.

[0112] Example 5

[0113] This embodiment illustrates that BI enhances the killing effect of T cells on gastric cancer cells by inhibiting the Wnt / β-catenin / PD-L1 signaling pathway.

[0114] (i) To investigate the regulatory effect of BI synergistic T cells on the expression levels of immune checkpoint-related proteins PD-L1 and PD-1.

[0115] Logarithmic growth phase MKN45 cells were seeded at 600 cells / well in a six-well plate. After cell attachment, four experimental groups were set up:

[0116] ① Control group: Only an equal volume of culture medium was added, without cell treatment;

[0117] ② BI group: Only BI was added to a final concentration of 100 μmol / L;

[0118] ③T cell group: T cells were added at a ratio of MKN45 cells to T cells of 1:5;

[0119] ④BI+T cell group: T cells were added at a ratio of MKN45 cells to T cells of 1:5, and BI was added at a final concentration of 100 μmol / L.

[0120] After incubation at 37°C and 5% CO2 for 72 hours, the expression of PD-L1, PD-1 and GAPDH proteins was detected by Western blot.

[0121] The results are as follows Figure 13 As shown, compared with the T cell group, BI group and control group, the BI combined with T cell group significantly inhibited PD-L1 protein expression (P<0.01), but had no significant effect on PD-1 protein expression (P>0.05).

[0122] (ii) To investigate the regulatory role of BI in the expression levels of key proteins in the Wnt / β-catenin signaling pathway by T cells.

[0123] After incubation at 37℃ and 5% CO2 for 72 h, the expression of Wnt / β-catenin signaling pathway-related proteins β-catenin, pGSK-3β, GSK-3β and GAPDH proteins was detected by Western blot.

[0124] The results are as follows Figure 14 As shown, compared with the T cell group, BI group and control group, the BI combined with T cell group significantly inhibited the expression of pGSK-3β and β-catenin proteins (P<0.01 or P<0.05), that is, it inhibited the Wnt / β-catenin signaling pathway.

[0125] (iii) To investigate whether BI enhances the killing effect of T cells on gastric cancer cells by regulating the Wnt / β-catenin pathway.

[0126] Logarithmic growth phase MKN45 cells were seeded at 600 cells / well in a six-well plate. After cell attachment, three experimental groups were set up:

[0127] ① Control group: Only an equal volume of culture medium was added, without cell treatment;

[0128] ②BI+T cell group: T cells were added at a ratio of MKN45 cells to T cells of 1:5, and BI was added at a final concentration of 100 μmol / L.

[0129] ③BI+T cell+LiCl group: MKN45 cells were pretreated with 50μmol / L GSK-3β activity inhibitor LiCl for 6h, T cells were added at a ratio of MKN45 cells to T cells of 1:5, and BI was added at a final concentration of 100μmol / L.

[0130] After incubation at 37℃ and 5% CO2 for 72 h, the expression of pGSK-3β, GSK-3β, PD-L1 and GAPDH proteins was detected by Western blot.

[0131] The results are as follows Figure 15 As shown, the addition of LiCl reversed the inhibitory effect of BI combined with T cells on pGSK-3β and PD-L1 proteins (P<0.01).

[0132] This example illustrates that the combination of β-ionone and T cells can enhance the anti-tumor immune response by inhibiting the Wnt / β-catenin signaling pathway and downregulating the expression of PD-L1 in tumor cells.

[0133] Example 6

[0134] This embodiment investigated the effect of BI on enhancing T cell activity and killing ability in vivo.

[0135] I. A xenograft tumor model was constructed by inoculating mouse forestomach cancer MFC cells into the right inguinal region of C57BL / 6 mice.

[0136] Four-week-old female C57BL / 6 mice were randomly divided into a control group and a BI group, with five mice in each group. Intraperitoneal administration began two days before vaccination. Mice in the BI group were injected intraperitoneally with BI at a dose of 50 mg / kg every other day, while mice in the control group were injected with an equal volume of 20% DMSO. The administration was repeated twice.

[0137] MFC cells in the logarithmic growth phase were collected and their concentration adjusted to 6.0 × 10⁻⁶ with PBS. 7 Cells / mL. 100 μL of cell suspension (containing 6.0 × 10⁶ cells / mL) was subcutaneously injected into the right groin of mice. 6 (cells). After inoculation, mice in the BI group and the control group were intraperitoneally injected with BI or an equivalent amount of 20% DMSO at a dose of 50 mg / kg every 1 day for 2 weeks. The weight of the mice and the volume of the xenograft were measured and recorded every 2 days.

[0138] The results are as follows Figure 16 As shown, the body weight of both groups of mice did not change significantly throughout the experiment, indicating that BI was essentially non-toxic to mice at the dosage used (P>0.05). Compared with the control group, the volume and weight of the xenograft in the BI group were significantly smaller than those in the control group (P<0.01), indicating that BI can inhibit the growth of gastric cancer in vivo.

[0139] 2. Collect peripheral blood from mice for blood cell analysis.

[0140] The experiment continued until day 17 when the mice were sacrificed, and the transplanted tumor tissue and peripheral blood were collected. The tissue was used for Western blot detection, and the peripheral blood was used for complete blood count and flow cytometry.

[0141] 100 μL of peripheral blood was collected from mice, and 5 μL each of mouse CD3, CD4, and CD8 fluorescently labeled flow cytometry antibodies were added sequentially. The mixture was incubated at 4°C in the dark for 30 min. 1 mL of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 5 min. The cells were then centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were washed once with PBS. The cells were resuspended in 500 μL of PBS. Detection was performed using a BD FACSCalibur flow cytometer, and CD3+ was analyzed using FlowJo software. + / CD4 + and CD3 + / CD8 + T cell ratio.

[0142] Peripheral blood cell count results as follows Figure 17 As shown, the peripheral blood white blood cell count and lymphocyte count in the BI group mice were significantly increased (P<0.05) and significantly increased (P<0.05). Flow cytometry results are as follows. Figure 18 As shown, CD8⁺ T cells and CD4⁺ T cells in the peripheral blood of mice in the BI group + The proportion of T cells also increased significantly. This indicates that BI has a clear immune-activating effect, which can enhance the body's cellular immune response by increasing the number of immune cells in peripheral blood and optimizing the proportion of T cell subsets.

[0143] This example demonstrates that β-ionone can inhibit gastric cancer growth in vivo by enhancing T cell activation and killing effects.

[0144] Example 7

[0145] This embodiment verifies that BI enhances the killing effect of T cells on gastric cancer cells in vivo by inhibiting the Wnt / β-catenin / PD-L1 signaling pathway.

[0146] Based on the mouse model experiment in Example 6, this embodiment uses Western blot to detect the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) proliferating cell nuclear antigen (PCNA), Wnt / β-catenin signaling pathway-related β-catenin, pGSK-3β, GSK-3β, PD-L1, PD-1 proteins and internal reference protein β-actin in the xenograft tissues of two groups of mice.

[0147] The results are as follows Figure 19 , Figure 20 and Figure 21 As shown, the BI group significantly inhibited the expression of PCNA, Cyclin D1, CDK4, β-catenin, pGSK-3β, and PD-L1 proteins (P<0.01 or P<0.05), but had no significant inhibitory effect on PD-1 (P>0.05). These results indicate that BI can not only inhibit tumor growth and cell proliferation in vivo, but also downregulate the activity of the Wnt / β-catenin signaling pathway and PD-L1 expression. This suggests that BI acts in vivo and in vitro in a consistent manner, reducing the proliferative capacity and immune escape potential of tumor cells by inhibiting the Wnt / β-catenin / PD-L1 signaling pathway, thereby enhancing the killing effect of T cells on gastric cancer cells.

Claims

1. The application of β-ionone in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator contains 0.05-99.5% β-ionone by mass.

2. The application of β-ionone according to claim 1 in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator also contains pharmaceutically acceptable excipients.

3. The application of β-ionone according to claim 2 in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator is an injectable formulation.

4. The application of β-ionone according to claim 3 in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator is a tumor immune checkpoint inhibitor, and the tumor immune checkpoint is PD-L1.

5. The application of β-ionone according to claim 4 in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator is a tumor immunosensitizer, and the tumor immunosensitization is mediated by T cells.

6. The use of β-ionone according to any one of claims 1-5 in the preparation of tumor immunomodulators, characterized in that, The tumor immunomodulator has at least one of the following uses: (1) Downregulate the expression of PD-L1 in tumor cells and weaken tumor immune escape; (2) Enhance CD8 + Antitumor activity of cytotoxic T cells; (3) Downregulates the activity of the Wnt / β-catenin signaling pathway in tumor cells; (4) Activate the immune response of the tumor microenvironment and synergistically enhance the killing effect of T cells.

7. The application of β-ionone according to claim 6 in the preparation of tumor immunomodulators, characterized in that, The tumor is stomach cancer.

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