Use of piceatannol as immune function enhancer in tumor immunotherapy

Abstract

The use of piceatannol as an immune function enhancer in tumor immunotherapy, which use belongs to the technical field of biomedicine. Piceatannol can regulate the ratio and function of immune cells such as T cells, B cells, macrophages, neutrophils, monocytes and myeloid-derived suppressor cells, thereby improving an immune microenvironment in the body, remodeling a tumor immune microenvironment by means of inhibiting the migration of immunosuppressive cells to tumors, and inhibiting the growth and metastasis of melanoma, colorectal cancer and breast cancer. Piceatannol has application prospects in the treatment and prevention of various cancers. It is suggested that piceatannol can significantly improve the effects on anti-tumor immune responses and remodel the tumor immune microenvironment, thereby effectively inhibiting tumor proliferation, growth and metastasis, and can be used for the preparation of a drug for regulating immune cell functions or can be used as a lead compound for the development of a tumor immunotherapeutic drug.

Description

Application of piceatannol as an immune function enhancer in tumor immunotherapy Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the application of piceatannol as an immune function enhancer in tumor immunotherapy. Background Art

[0002] Breast cancer is a benign or malignant tumor caused by abnormal division and proliferation of mammary epithelial cells. Globally, breast cancer ranks second in incidence, accounting for 11.6% of all cancers, and has a mortality rate of 6.6%. Despite recent advances in diagnosis and treatment, breast cancer remains a global health problem affecting millions of people.

[0003] Common treatments for breast cancer include chemotherapy, surgery, radiotherapy, and hormone therapy. However, these treatments can cause adverse reactions and multidrug resistance, leading to complications. Natural products are abundant and diverse, with multi-target mechanisms of action and a favorable safety profile. Therefore, there is a growing demand for new anticancer drugs derived from medicinal plants and natural products.

[0004] Summary of the Invention

[0005] The purpose of the present application is to provide the use of piceatannol as an immune function enhancer in tumor immunotherapy. The piceatannol can be used to prepare a regulator for regulating the ratio and / or function of immune cells or as a lead compound for the development of tumor immunotherapy drugs.

[0006] The present application provides the use of piceatannol in preparing an immune cell ratio and / or function regulator.

[0007] Preferably, the immune cells include at least one of the following: T cells, B cells, macrophages and bone marrow-derived immunosuppressive cells.

[0008] Preferably, the regulator has at least one of the following effects: (1) increasing the ratio of T / B immune cells of lymphoid origin and enhancing the body's anti-tumor killing ability;

[0009] (2) Inhibit the ratio of myeloid-derived cells, reduce the promoting effect of neutrophils and myeloid-derived suppressor cells on tumor growth and metastasis, and the inhibitory effect on anti-tumor T cell responses;

[0010] (3) Inhibit immunosuppressive reactions and promote anti-tumor immune responses;

[0011] (4) Activate B cells and relieve some immune suppression;

[0012] (5) Reduce the number of immunosuppressive cells in tumors, reshape the immune microenvironment, and enhance the body's anti-tumor immune response.

[0013] Preferably, the regulatory effect of the immune cell ratio regulator includes increasing the cell ratio of T cells, CD8+T cells and B cells; and reducing the cell ratio of macrophages, neutrophils, monocytes and myeloid-derived suppressor cells.

[0014] The present application also provides the use of piceatannol in the preparation of an immune enhancer.

[0015] The present application also provides the use of piceatannol in the preparation of tumor immunotherapy drugs.

[0016] Preferably, the effects of the tumor immunotherapy drug include at least one of the following: (1) inhibiting immunosuppressive cells;

[0017] (2) Inhibit the growth and migration of tumor cells;

[0018] (3) Enhance the ability of immune cells to kill tumor cells.

[0019] Preferably, the tumor includes breast cancer, melanoma and colorectal cancer.

[0020] Preferably, the piceatannol has a structure shown in Formula I:

[0021] Preferably, the piceatannol is extracted from passion fruit.

[0022] Beneficial effects: This application uses piceatannol to conduct in vivo experiments on MMTV-PyVT transgenic mice with spontaneous breast cancer, C57 tail vein injection melanoma model, and colorectal cancer PDX model mice, and evaluates the proportion and functional activity of immune cells in the spleen and tumor of mice. It is proved that piceatannol can regulate the proportion and function of immune cells such as T cells, B cells, macrophages, neutrophils, monocytes, and myeloid-derived suppressor cells, thereby improving the body's immune microenvironment, and reshape the tumor immune microenvironment by inhibiting the migration of immunosuppressive cells to tumors, inhibiting the growth and metastasis of melanoma, colorectal cancer and breast cancer, and has application prospects in treating and preventing various cancers. It shows that piceatannol can significantly enhance the anti-tumor immune response and reshape the tumor immune microenvironment, thereby effectively inhibiting tumor proliferation, growth and metastasis, and can be used to prepare drugs that regulate immune cell functions or as a lead compound for the development of tumor immunotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a HPLC analysis chart of piceatannol reference substance;

[0024] FIG2 is an HPLC analysis chart of piceatannol extracted from passion fruit according to the present invention;

[0025] FIG3 is a graph showing the effect of piceatannol on the number of immune cells in the spleen microenvironment;

[0026] FIG4 is a graph showing the effect of piceatannol on the expression of the S100a8 gene in the spleen immune cell subset;

[0027] FIG5 is a graph showing the effect of piceatannol on the expression of S100a9 gene in spleen immune cell subsets;

[0028] FIG6 is a graph showing the effect of piceatannol on the expression of Mmp9 gene in spleen immune cell subsets;

[0029] FIG7 is a graph showing the effect of piceatannol on the expression of Ifitm3 gene in spleen immune cell subsets;

[0030] FIG8 is a graph showing the effect of piceatannol on the expression of Wfdc17 gene in spleen immune cell subsets;

[0031] FIG9 is a graph showing the effect of piceatannol on Hmox1 gene expression in spleen immune cell subsets;

[0032] FIG10 is a graph showing the effect of piceatannol on the expression of Csflr gene in spleen immune cell subsets;

[0033] FIG11 is a graph showing the effect of piceatannol on the number of immunosuppressive CD45+CD4+Foxp3+Treg cells in the spleen of mice;

[0034] FIG12 is a graph showing the effect of piceatannol on the number of B cells in the spleen of mice;

[0035] FIG13 is a graph showing the effect of piceatannol on the number of immunosuppressive cells (MDSCs) in mouse tumors;

[0036] FIG14 is a graph showing the inhibitory effect of piceatannol on tumor T cell activity;

[0037] FIG15 is a diagram showing the effect of piceatannol on the expression of surface receptors and their main ligands of myeloid-derived suppressor cells;

[0038] FIG16 is a graph showing the effect of piceatannol on the expression of neutrophil elastase NE, CAMP protein, myeloperoxidase MPO, PADI4 protein, and cathepsin G in mouse spleen tissue;

[0039] FIG17 is a graph showing the effect of piceatannol on the expression of neutrophil elastase (NE,elane) and matrix metalloproteinase 9 (MMP9);

[0040] FIG18 is a graph showing the effect of piceatannol on the changes in tumor volume in mice;

[0041] FIG19 is a graph showing the effect of piceatannol on the body weight of mice;

[0042] FIG20 is a graph showing the effect of piceatannol treatment on tumors;

[0043] FIG21 is a graph showing the effect of piceatannol on lung metastasis of breast cancer;

[0044] FIG22 is a graph showing the effect of piceatannol on tumor growth in colorectal cancer PDX model mice;

[0045] FIG23 is a graph showing the effect of piceatannol on body weight changes in colorectal cancer PDX model mice;

[0046] FIG24 is a graph showing the effects of piceatannol on tumor changes in colorectal cancer PDX model mice;

[0047] FIG25 is a graph showing the effect of piceatannol on melanoma lung metastasis. DETAILED DESCRIPTION

[0048] The present application provides the use of piceatannol in preparing an immune cell ratio and / or function regulator.

[0049] The piceatannol described in the present application preferably has a structure shown in Formula I:

[0050] The present application does not specifically limit the source of the piceatannol. In the examples, piceatannol extracted from passion fruit is used as an example. According to the article The dried and milled seeds were extracted in ethanol. Then the extracts were combined, filtered and concentrated. The final seed extract was extracted by ethyl acetate (EtOAc). The EtOAc extract was subjected to a silica gel column (PE / EtOAc, 100:1 to 0:1) and eight fractions obtained. The fraction Fr.3 was subjected to Sephadex LH-20 gel with methanol as eluent then semi-preparative HPLC (acetonitrile / H2O, 5:95 to 1:0) to obtain purified PIC., the present application separates the piceatannol from passion fruit, preferably comprising the following steps: (1) crushing the dried seeds of passion fruit and extracting them with ethanol, recovering the ethanol and evaporating it until there is no alcohol taste to obtain an extract;

[0051] (2) mixing the extract with distilled water, and extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extract;

[0052] (3) The ethyl acetate extract was separated by normal phase silica gel column chromatography and eluted with petroleum ether-ethyl acetate gradient (petroleum ether / ethyl acetate v / v, 50:1, 10:1, 4:1, 2:1, 1:2, 1:4, 0:1). The extract was detected by thin layer chromatography and similar components were combined to obtain 8 components Fr.1-Fr.8;

[0053] (4) Fraction Fr.3 from step (3) was separated by Sephadex LH-20 column chromatography using isocratic elution with methanol, followed by semi-preparative chromatography (acetonitrile / water (v / v) = 5 / 95 from 0 to 50 min; acetonitrile / water (v / v) = 60 / 40 from 50 to 60 min; acetonitrile / water = 100 / 0 from 50 to 60 min) to obtain piceatannol.

[0054] The piceatannol described in the present application has the effect of regulating the ratio and function of immune cells, preferably including at least one of the following: (1) increasing the ratio of lymphoid-derived T / B immune cells and enhancing the body's anti-tumor killing ability; (2) inhibiting the ratio of myeloid-derived cells, reducing the promoting effect of neutrophils and bone marrow-derived suppressor cells on tumor growth and metastasis and the inhibitory effect on anti-tumor T cell response; (3) effectively inhibiting immunosuppressive reactions and promoting anti-tumor immune responses; (4) activating B cells and relieving part of immunosuppression; (5) reducing the number of immunosuppressive cells in tumors to reshape the immune microenvironment and enhance the body's anti-tumor immune response. The immune cells described in the present application preferably include at least one of the following: T cells, B cells, macrophages and bone marrow-derived immunosuppressive cells, wherein bone marrow-derived immunosuppressive cells include neutrophils and monocytes, and the regulation preferably includes: increasing the cell ratio of T cells, CD8+T cells and B cells in mouse spleen immune cells; while reducing the cell ratio of macrophages, neutrophils, monocytes and bone marrow-derived suppressor cells in mouse spleen and tumor immune cells.

[0055] The present application also provides the use of piceatannol in the preparation of an immune enhancer.

[0056] The leucopeptide described in this application can significantly increase T cells (especially CD8+ T cells) and B cells, while reducing the number of macrophages, neutrophils, monocytes, and bone marrow-derived immunosuppressive cells and inhibiting their function, affecting the migration of immunosuppressive cells to tumors and the remodeling of the tumor extracellular matrix. This shows that leucopeptide can significantly enhance the anti-tumor immune response and reshape the tumor immune microenvironment, thereby effectively inhibiting tumor proliferation, growth, and metastasis.

[0057] The present application also provides the use of piceatannol in the preparation of tumor immunotherapy drugs.

[0058] The tumors described in the present application preferably include breast cancer, melanoma and colorectal cancer.

[0059] To further illustrate the present application, the application of piceatannol provided in the present application as an immune function enhancer in tumor immunotherapy is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present application.

[0060] Example 1

[0061] Isolation, preparation and identification of piceatannol:

[0062] 1. Separation and preparation:

[0063] Step 1: Crush 350g of dried passion fruit seeds, add 35L of 80% ethanol by volume, stir well, and repeatedly reflux extract 3 times, each time for 3 hours. Filter, combine the filtrate, recover the ethanol under reduced pressure and evaporate until there is no alcohol taste to obtain 52.5g of extract.

[0064] Step 2: Dissolve and disperse the extract from step 1 in 2 L of distilled water, then extract with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain 41 g of the ethyl acetate fraction.

[0065] Step 3: The ethyl acetate fraction from step 2 was separated by normal phase silica gel (100-200 mesh, Qingdao Ocean Chemical Plant) column chromatography, and gradient eluted with petroleum ether-ethyl acetate (100:0, 50:1, 10:1, 2:1, 1:1, 1:2, 1:4, 0:1) in sequence. The fraction was detected by thin layer chromatography (TLC), and the components were observed for similarity by color development with 10% (v / v) sulfuric acid-ethanol. Similar components were combined to obtain 8 components Fr.1 to Fr.8.

[0066] Step 4: 3.5 g of Fr.3 (i.e., the fraction eluted with petroleum ether and ethyl acetate in a ratio of 1:1) was subjected to Sephadex LH-20 (Cytiva) column chromatography with isocratic elution using methanol, and the middle fraction (the fraction collected after approximately 10 to 16 h) was collected. Finally, piceatannol was obtained by semi-preparative chromatography (Shimadzu LC-20A, SPD-M40 diode array detector, detection wavelength of 254 nm, YMC Pack ODS-A, 5 μm, 10 mm × 250 mm) using a water-acetonitrile mobile phase gradient elution (acetonitrile / water (v / v) = 5 / 95 from 0 to 50 min; acetonitrile / water (v / v) = 60 / 40 from 50 to 60 min; acetonitrile / water = 100 / 0 from 50 to 60 min).

[0067] Comparison of HPLC results between the piceatannol reference substance ( FIG. 1 ) and the purified piceatannol ( FIG. 2 ) revealed that the retention times of the two substances were consistent under the same chromatographic conditions.

[0068] 2. Identification of Piceatannol

[0069] 1 H-NMR and 13 The C-NMR spectrum was measured using a 600 MHz NMR spectrometer with DMSO-d6 as the internal standard; the mass spectrometry data were measured using a Shimadzu IT-TOF mass spectrometer. 1H-NMR spectrum signals are: 6.11 (1H, t, J = 2.4 Hz), 6.37 (2H, d, J = 2.4 Hz), 6.71 (1H, d, J = 8.4 Hz), 6.72 (1H, d, J = 16.2 Hz), 6.84 (1H, dd, J = 8.4, 1.8 Hz), 6.85 (1H, d, J = 16.2 Hz), 6.96 (1H, d, J = 1.8 Hz); 13 C-NMR spectrum signals are 158.94, 145.97, 145.85, 139.70, 129.07, 128.69, 125.98, 119.03, 116.14, 113.75, 104.74, 102.17; MS (ESI) m / z: 243.07 [MH] - In summary, the compound was identified as piceatannol. Mathi et al. have reported piceatannol in the literature. 1 H-NMR and 13 C-NMR and other related data, references are as follows [1] Mathi P, Das S, Nikhil K, et al. Isolation and Characterization of the Anticancer Compound Piceatannol from Sophora Interrupta Bedd[J]. International Journal of Preventive Medicine, 2015, 6(1). DOI: 10.4103 / 2008-7802.167181.

[0070] Example 2

[0071] Piceatannol regulates the number and function of immune cells in mice and enhances their anti-tumor immune response.

[0072] 1. Experimental methods:

[0073] (1) Single-cell transcriptome sequencing technology detects changes in the number and function of immune cells in mouse spleens

[0074] After sample separation, the standard 10x Genomics sample preparation method was used. The tissue sample was first cut into small pieces, then digested with collagenase and incubated in a shaker at 37°C. The cell suspension after digestion and incubation was filtered through a strainer, and the filtered contents were centrifuged to remove the enzyme. The supernatant was taken to count the cells, and the cell density was adjusted based on the cell count results, and then single-cell sequencing was performed. Single-cell sequencing used the 10×Genomics Single Cell A Chip Kit. The target cell recovery number was 10,000, and the viability was greater than 80%. The sequencing company was commissioned to perform single-cell sequencing. The genes of the collected sample cells were clustered and classified according to the graph-based clustering algorithm. The cells within the same cluster had the closest expression patterns.

[0075] (2) Flow cytometry detection of changes in the proportion of lymphocytes in mouse spleen

[0076] After killing the mice, the spleen was removed by sterile laparotomy and ground. After filtering through a 200-mesh filter, the pellet was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, 1 mL of red blood cell lysis buffer was added, the pellet was allowed to stand at room temperature for 2 min, and 9 mL of normal saline was added to terminate the lysis. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 0.5 × 10 8 cells / mL to 1.0×10 8 pieces / mL.

[0077] A PBS solution containing 0.5% fetal bovine serum was prepared. The corresponding antibodies and 100 μL of cell suspension were added to 1 mL of PBS containing 0.5% fetal bovine serum according to the ratio. The cells were incubated at 4°C in the dark for 30 minutes. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded and the tubes were washed once with PBS. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in 400 μL of PBS containing 0.5% fetal bovine serum per tube. The cells were analyzed by flow cytometry. Changes in the proportions of Treg cells (CD45+CD4+Foxp3+) and B cells (CD45+CD3-CD19+) in the spleen were measured by flow cytometry.

[0078] (3) Flow cytometry detection of the proportion of immunosuppressive cells in mouse tumors

[0079] Isolate the tumor tissue, mince the tissue with curved scissors, add enough Digestion Buffer (1640 medium + 10% FBS + 0.5 mg / mL Collagenase D + 0.1 mg / mL DNaseI), and shake on a shaker for 1 hour. Pour the digested tumor tissue onto a 100-mesh sieve, grind it, pass it through a 200-mesh sieve, and centrifuge it at 1600 rpm for 5 minutes. Resuspend it with Ficoll, overlay serum-free 1640 medium on top, and centrifuge it at 800g for 20 minutes. Aspirate the middle layer containing tumor-associated immune cells, centrifuge it at 1600 rpm for 5 minutes. Resuspend it with 1 mL FACS and count it on a cell counting plate. Stain it with CD45-PE-Cy7, CD11b-FITC, Ly6C-PE, and Ly6G-PerCP antibodies for 30 minutes. After termination, pass it through a 40 μM sieve and detect it by flow cytometry.

[0080] (4) qPCR detection of changes in mouse tumor microenvironment

[0081] 1) Total RNA extraction: Mouse spleen and tumor tissue were homogenized in Trizol and digested using a tissue homogenizer. Add 0.25 mL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes, transfer the upper aqueous phase to another Eppendorf tube, add an equal volume of isopropanol, mix well, and let stand at -20°C for 20 minutes. Centrifuge at 10,000 rpm at 4°C for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol, vortex vigorously to wash the precipitate, centrifuge at 6,000 rpm at 4°C for 5 minutes, and discard the supernatant. Repeat steps 5 and 6, and air-dry the precipitate at room temperature. Dissolve the precipitate in RNase-free water and measure the RNA concentration.

[0082] 2) Reverse Transcription: 1000 ng of the total RNA sample was thoroughly mixed with 4 μL of 5X Reverse Transcription Buffer and RNase-free water, and reverse transcription was performed. cDNA was obtained using the 5X All-In-One RT MasterMix Kit according to the kit instructions.

[0083] 3) Perform PCR amplification on the cDNA template: 1 μL of the cDNA sample was thoroughly mixed with the qPCR mixture and then amplified using Vazyme Taq Pro Universal SYBR qPCR Master Mix reagent, following the kit instructions.

[0084] Table 1 Primers used for PCR amplification

[0085] (5) Mass spectrometry detection of changes in mouse serum proteome

[0086] Collect mouse blood into a vacuum tube, mix thoroughly by inverting, incubate at room temperature for 45 minutes, and centrifuge at 3000 rpm for 10 minutes at 4°C. Transfer serum to a centrifuge tube, add protease inhibitors, mix thoroughly, centrifuge, and aliquot samples for subsequent proteomic analysis.

[0087] 1) Protein sample preparation

[0088] Add an appropriate amount of pre-cooled acetone to the above serum and shake it to mix it thoroughly, then place it at -20℃ overnight to precipitate. Centrifuge it the next day at 4℃ and 12000g for 10 minutes, discard the supernatant, and retain the precipitate at the bottom of the tube. Take an appropriate amount of pre-cooled acetone and add it to the above precipitate, place the mixture in a vortex and shake it to mix it thoroughly, centrifuge it at 4℃ and 12000g for 15 minutes, discard the supernatant and collect the precipitate. Repeat this step once. Place the precipitate obtained above in a fume hood to dry at room temperature, add an appropriate amount of tissue lysis buffer, shake it with a vortex to mix it, and fully lyse the protein. Centrifuge it at 12000g at room temperature for 15 minutes, take the supernatant and transfer it to a new EP tube.

[0089] 2) Detect protein concentration

[0090] The protein sample was taken out and placed on ice. BCA working solution was prepared according to the instructions of the BCA kit, with 200 μL prepared for each well.

[0091] Prepare an appropriate amount of 0.5 mg / mL protein standard solution according to the BSA instructions. Then, add the protein standard and diluent to the standard wells of a 96-well plate according to the corresponding dilution factor. To improve accuracy, set up two sets of replicate wells. Add 2 μL of the sample to be tested to each sample well, and add 18 μL of the standard diluent to each well, for a total of 20 μL. Only after the addition of the above reagents is completed can the BCA working solution be added, 200 μL per well, and then the 96-well plate with the added samples is placed at 37°C and incubated for 30 minutes. Use a microplate reader to measure the absorbance value of each well at 562 nm. Use the concentration of the standard well and the corresponding OD value to create a standard curve. Then use the OD value in each sample well to calculate the protein concentration.

[0092] 3) SDS-PAGE electrophoresis

[0093] Keep samples on ice throughout the entire process. Add the appropriate amount of 3× loading buffer based on the sample's protein concentration and volume, mix thoroughly, and place in a 100°C metal bath for 5 minutes to completely denature. Prepare a 12% SDS-PAGE gel and perform electrophoresis on 10 μg of each sample. Set the electrophoresis voltage to 70V for the stacking gel and 120V for the separating gel, then run the gel under the set constant voltage. After electrophoresis, open the glass plate, gently remove the gel, and excise the redundant gel above and below. Stain the remaining gel with Coomassie Brilliant Blue. Wash with water until the background is clear. Scan the gel with an ImageScanner scanner after washing.

[0094] 4) Protein reductive alkylation and enzymatic hydrolysis

[0095] After protein quantification, 100 μg of each sample was taken, 5 volumes of pre-cooled acetone were added to each sample, and the samples were placed at -20°C for 1 hour to fully precipitate the protein.

[0096] The mixture was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and the bottom precipitate was vacuum-frozen and dried. Dissolution Buffer was added, and after the protein was fully dissolved, 4 μL of Reducing Reagent was added. The mixture was then incubated at 60°C for 1 hour. Cysteine-Blocking Reagent was added to the reaction mixture, and the reductive alkylation reaction was carried out at room temperature. After the reaction, the protein solution was transferred to an ultrafiltration tube and centrifuged at 12,000 rpm for 25 minutes. 100 μL of Dissolution Buffer was added to the collected ultrafiltrate, and after thorough mixing, the mixture was centrifuged at 12,000 rpm for 20 minutes. This step was repeated three times. Sequencing-grade trypsin solvent was added to the collected solution and allowed to react fully. Dissolution Buffer was then added to the collected peptides, and the mixture was centrifuged and the bottom solution was collected.

[0097] 5) Protein labeling

[0098] Remove the iTRAQ reagent and add a predetermined amount of isopropanol to the sample according to the manufacturer's instructions. Once fully dissolved, proceed with the following steps. Add 50 μL of each reductively alkylated and enzymatically digested protein sample to the iTRAQ reagent and mix thoroughly. Incubate the mixture on a shaker at room temperature for 2 hours. Terminate the reaction by adding 100 μL of distilled water. Vortex to thoroughly mix, then centrifuge to the bottom of the tube. Freeze-dry the treated samples under vacuum and store until ready for use.

[0099] 6) LC-MS / MS analysis

[0100] Each dried histone sample was dissolved in mobile phase A and thoroughly mixed using a vortex shaker. Peptide separation experiments were performed on this mixture using an Agilent 1200 HPLC instrument. Following peptide separation, mass spectrometry identification and analysis were performed using a TripleTOF 5600 system (AB SCIEX) and a Nanospray III ion source (AB SCIEX).

[0101] 7) Protein qualitative, quantitative and identification

[0102] After obtaining the raw LC-MS / MS data, the raw files were analyzed using MaxQuant (version 1.6) and the corresponding proteins were identified by comparison with the Swiss-Prot human protein sequence database. At the protein and peptide levels, the false discovery rate of proteins was less than 1% (FDR < 1%), and at least two peptides were identified for further data processing.

[0103] 2. Experimental Results

[0104] (1) Effect of piceatannol on the number of immune cells in the spleen microenvironment

[0105] Single-cell cluster analysis of spleens from MMTV-PyVT mice (purchased from Southern Model Organisms) treated with piceatannol and controls yielded 15 subpopulations. As shown in Figure 3, after piceatannol treatment, subpopulations with increased cell proportions included T cells (marked by Trbc2 and cd3g), CD8+ T cells (marked by cd8b1 and cd3g), and B cells (marked by Ly6d and cd79). Meanwhile, the proportions of macrophages (marked by Hmox1 and C1qa), neutrophils (marked by Camp and S100a8 / 9), monocytes (marked by Lyz2 and Ifitm3), and myeloid-derived suppressor cells (marked by Wfdc17 and Il1b) were significantly reduced. This indicates that piceatannol effectively increases the ratio of T / B immune cells of lymphoid origin, enhances the body's anti-tumor killing ability, while inhibiting the ratio of myeloid-derived cells, reducing the promoting effect of neutrophils and myeloid-derived suppressor cells on tumor growth and metastasis, and the inhibitory effect on anti-tumor T cell response.

[0106] (2) Effects of piceatannol on gene expression of splenic immune cell subsets

[0107] The results, as shown in Figures 4 and 5, showed a significant decrease in the expression of S100A8 and S100A9 genes, which were positively correlated with neutrophil infiltration and significantly enriched in pathways involved in neutrophil activation, migration, and chemotaxis, indicating that piceatannol can reduce the ability of neutrophils to migrate toward tumor cells. Furthermore, downregulation of S100A9 in myeloid-derived suppressor cells indicated that the migration and activation of MDSCs were inhibited, thereby reducing their impact on CD8+ T cell proliferation. Suppression of Mmp9 (Figure 6) further reduced the likelihood of tumor angiogenesis. Decreased expression of Ifitm3 (Figure 7), a gene involved in inflammatory activation pathways, in monocytes suggests a reduction in inflammatory responses and a remodeling of the immune environment. Downregulation of the immunosuppressive gene Wfdc17 (Figure 8) and the macrophage M2-associated genes Hmox1 (Figure 9) and Csf1r (Figure 10) suggests that piceatannol can effectively suppress immunosuppressive responses and promote anti-tumor immune responses.

[0108] (3) Effect of piceatannol on the number of immune cells in mouse spleen

[0109] Flow cytometry results, as shown in Figure 11, show that piceatannol significantly decreased the number of immunosuppressive CD45+CD4+Foxp3+Treg cells in the spleen, while increasing the number of B cells (Figure 12). This suggests that piceatannol may have a certain anti-tumor effect by activating B cells and relieving some immunosuppression.

[0110] (4) Effect of piceatannol on the number of immunosuppressive cells in mouse tumors

[0111] The flow cytometry results are shown in Figure 13. Piceatannol significantly reduced the number of immunosuppressive cells MDSC cells in the tumor microenvironment, indicating that piceatannol reshapes the immune microenvironment by reducing the number of immunosuppressive cells in the tumor, thereby enhancing the body's anti-tumor immune response and exerting its anti-tumor effect.

[0112] (5) Effects of piceatannol on the tumor immune microenvironment and spleen anti-tumor immune response in mice

[0113] qPCR assays were used to examine the expression of genes associated with the suppression of T cell activity. As shown in Figure 14, the expression of TOX, a protein associated with exhaustion of tumor-infiltrating T cells, and Pdcd1, an inhibitory receptor for T cell antigen activation, were significantly decreased, indicating that piceatannol inhibits tumor-induced T cell activity. Furthermore, the expression of CXCR2, a surface receptor for myeloid-derived suppressor cells (MDSCs), and its major ligands CXCL1, CXCL2, CXCL3, and CXCL5 were significantly downregulated (Figure 15), suggesting that the ability of MDSCs to migrate into the tumor microenvironment is impaired, thereby impacting their ability to suppress anti-tumor immune responses. Serum proteomic analysis revealed a 40% decrease in histone H2A, a protein involved in neutrophil extracellular trap formation. Furthermore, the expression of neutrophil elastase NE, CAMP protein, myeloperoxidase (MPO), PADI4 protein, and cathepsin G in mouse spleen tissue was downregulated by approximately 80% (Figure 16), indicating that reduced neutrophil extracellular trap formation, in turn, attenuates the neutrophil-promoting effect on tumor survival and metastasis. In the tumor microenvironment, two proteases closely related to tumor metastasis, neutrophil elastase (NE,elane) and matrix metalloproteinase 9 (MMP9), were significantly downregulated under the action of piceatannol, affecting the remodeling of the extracellular matrix and ultimately inhibiting tumor metastasis (Figure 17).

[0114] The above results show that piceatannol can not only enhance the anti-tumor function of immune cells, but also inhibit the function and recruitment of immunosuppressive cells, improve the tumor microenvironment, and enhance the immunotherapy effect of breast cancer.

[0115] Example 3

[0116] Effects of piceatannol on growth and metastasis of breast cancer

[0117] 1. Experimental methods:

[0118] (1) Measurement of tumor volume and body weight changes in each group of experimental animals

[0119] Twelve female MMTV-PyVT mice, 7 weeks old, were housed in a controlled environment with a relative humidity of 45-55%, a controlled temperature of 20-25°C, and a 12-hour light / 12-hour dark cycle. The mice were randomly divided into two groups (6 mice / group): a control group and a piceatannol group (80 mg / kg). The control group received 0.4 mL of distilled water orally, while the piceatannol group received 80 mg / kg of piceatannol solution once daily for 30 days. Tumor size and body weight were measured every three days. At the end of the experiment, all mice were euthanized, and blood, tumor, lung, and spleen samples were collected for analysis.

[0120] (2) HE staining to detect lung metastasis in MMTV-PyVT breast cancer model mice

[0121] The mouse lungs were rinsed clean and placed in 4% paraformaldehyde solution for 24 hours, then dehydrated, embedded in paraffin, and sectioned. The sections were stained with hematoxylin-eosin for 5 minutes, dehydrated, and mounted with neutral gum.

[0122] 2. Experimental results:

[0123] (1) Piceatannol significantly inhibited the growth of breast cancer tumors in MMTV-PyVT transgenic mice, with an inhibition rate of 67%. Figures 18 and 19 show that compared with the control group (con), the tumor volume of mice in the piceatannol group (PIC) was significantly reduced, while there was no significant difference in body weight (Figure 20).

[0124] (2) Effect of piceatannol on lung metastasis of breast cancer

[0125] The HE staining results of the mouse lung tissue are shown in Figure 21: the control group had visible metastatic tumors in the lungs. After intervention with piceatannol, the size and number of the metastatic tumors were significantly reduced, demonstrating that piceatannol has an inhibitory effect on lung metastasis of breast cancer.

[0126] Example 3

[0127] Effects of piceatannol on the growth of colorectal cancer

[0128] 1. Experimental methods:

[0129] Measurement of tumor volume and body weight changes in each group of experimental animals

[0130] Eight male NOD-SCID mice, 7 weeks old, were housed in an environment with a relative humidity of 45-55%, a controlled temperature of 20-25°C, and a 12-hour light / 12-hour dark cycle. Fresh colorectal tumor tissue was obtained and cut into small pieces. The mice were inoculated on the ventral side and observed daily for tumor formation. Once tumors were observed, the mice were randomly divided into two groups (4 mice / group): a control group and a piceatannol group (80 mg / kg). The control group received 0.4 mL of distilled water by gavage, while the piceatannol group received an aqueous solution of piceatannol once daily for 13 weeks. Tumor size and body weight were measured every three days.

[0131] 2. Experimental results:

[0132] Piceatannol significantly inhibited tumor growth in colorectal cancer PDX model mice, achieving a tumor inhibition rate of 56%. The experimental results, shown in Figures 22 and 23, show that compared with the control group, the tumor volume of the piceatannol-treated mice was significantly reduced. There was no significant change in body weight in the control and piceatannol-treated mice (Figure 24).

[0133] Example 4

[0134] Effects of piceatannol on melanoma lung metastasis:

[0135] 1. Experimental methods:

[0136] Ten female C57BL / 6 mice, 6 weeks old, were housed in a controlled environment with a relative humidity of 45-55%, a controlled temperature of 20-25°C, and a 12-h light / 12-h dark cycle. The mice were randomly divided into two groups (5 mice / group): a control group and a piceatannol group (80 mg / kg). The control group mice were gavaged with 0.4 mL of distilled water, while the piceatannol group mice were gavaged with the piceatannol aqueous solution once daily for 4 consecutive weeks. Starting from the second week of administration, each mouse was injected intravenously with 1×10 6 melanoma cells were cultured for 2 weeks and the metastasis of melanoma cells in the lungs was observed.

[0137] 2. Experimental results:

[0138] The results of the mouse lung tissue are shown in Figure 25: the control group mice had visible melanoma metastases in the lungs. After intervention with piceatannol, the size and number of metastatic tumors were significantly reduced, demonstrating that piceatannol has a significant inhibitory effect on melanoma lung metastasis.

[0139] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. Use of piceatannol in the preparation of immune cell ratio and / or function regulators.

2. The application according to claim 1, characterized in that: The immune cells include at least one of the following: T cells, B cells, macrophages and bone marrow-derived immunosuppressive cells.

3. The application according to claim 1, characterized in that: The regulator includes an agent having at least one of the following effects: (1) increasing the ratio of T / B immune cells of lymphoid origin and enhancing the body's anti-tumor killing ability; (2) Inhibit the ratio of myeloid-derived cells, reduce the promotion of tumor growth and metastasis by neutrophils and myeloid-derived suppressor cells, and reduce the inhibitory effect on anti-tumor T cell responses; (3) Inhibit immunosuppressive responses and promote anti-tumor immune responses; (4) Activate B cells and relieve some immune suppression; (5) Reduce the number of immunosuppressive cells in tumors, reshape the immune microenvironment, and enhance the body's anti-tumor immune response.

4. The use according to claim 1, characterized in that: The regulatory effect of the immune cell ratio regulator includes: increasing the cell ratio of T cells, CD8+T cells and B cells; and reducing the cell ratio of macrophages, neutrophils, monocytes and bone marrow-derived suppressor cells.

5. An immune cell ratio and / or function regulator, characterized in that The active ingredient is piceatannol.

6. Application of piceatannol in the preparation of immune enhancers.

7. An immune enhancer, characterized in that: The active ingredient is piceatannol.

8. Application of piceatannol in the preparation of tumor immunotherapy drugs.

9. The use according to claim 8, characterized in that: The effects of the tumor immunotherapy drug include at least one of the following: (1) inhibiting immunosuppressive cells; (2) Inhibit the growth and migration of tumor cells; (3) Enhance the ability of immune cells to kill tumor cells.

10. The use according to claim 8, characterized in that: The tumor includes breast cancer, melanoma or colorectal cancer.

11. A tumor immunotherapy drug, characterized in that: The active ingredient is piceatannol.

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