Use of shibulian in the treatment of anaplastic thyroid cancer

Abstract

The application discloses a use of shiblancin in resisting anaplastic thyroid cancer, the shiblancin resists growth of anaplastic thyroid cancer cells in vivo, and the shiblancin promotes M1 macrophage polarization to inhibit anaplastic thyroid cancer progression by regulating spleen tyrosine kinase. The application finds a brand-new action path of the shiblancin and a new indication of anaplastic thyroid cancer, and provides a new path for treatment of the anaplastic thyroid cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of Dendrobium oryzae extract in the treatment of undifferentiated thyroid cancer. Background Technology

[0002] Thyroid cancer is the most common endocrine malignancy, and its incidence is increasing year by year. Anaplastic thyroid cancer (ATC) is the most malignant type of thyroid cancer, often accompanied by extrathyroidal invasion or metastasis. It accounts for approximately 2% of all thyroid cancers, but its mortality rate accounts for 50% of all thyroid cancer-related deaths, and there is currently no effective treatment. Therefore, identifying the mechanisms of ATC occurrence and development, as well as potential therapeutic targets, is a current research hotspot and challenge.

[0003] Exploring therapeutic targets for tumors is a focus of attention for scholars both domestically and internationally. Tumor-associated macrophages (TAMs), as the most numerous immune cells infiltrating the tumor microenvironment (TME), play a crucial role in the occurrence and development of various malignant tumors and are considered important targets for cancer treatment. Currently, anti-tumor therapeutic strategies targeting TAMs mainly focus on three directions: TAM clearance, inhibition of TAM recruitment, and regulation of TAM polarization. Regulation of TAM polarization, such as that of natural products like paclitaxel and echinacea polysaccharides, has been shown to promote M1 polarization of TAMs, activate anti-tumor immunity, and inhibit tumor progression. Previous studies have shown that TAMs accumulate in large quantities in ATC cell lines, but the regulatory mechanism remains unclear and requires further investigation.

[0004] Targeted regulation of tumor markers (TAMs) has become a novel strategy in cancer immunotherapy. Current research has found that an increasing number of natural products can play an important role as immunomodulators in regulating TAM polarization. Therefore, finding new drugs among natural products that can regulate TAMs can fill the gap in natural drugs used in tumor immunotherapy and provide more drug options for clinical cancer treatment. Dendrobium is a perennial evergreen herbaceous plant belonging to the genus Dendrobium in the Orchidaceae family. It is mainly distributed in Australia, Europe, and Asia, and in China, it is mainly distributed in the southwestern and southern provinces. It is an important component in traditional Chinese medicine prescriptions for adjuvant cancer treatment. Moscatilin is a synergistic substance extracted from the rhizomes of plants in the genus Dendrobium in the Orchidaceae family. As one of the important active ingredients of Dendrobium, it has significant antitumor, antioxidant, anti-inflammatory, and neuroprotective activities. In recent years, its research in the field of antitumor has attracted much attention. Current research shows that the antitumor effect of moscatilin is mainly achieved by regulating tumor cells. Although dendrobium oryzae has been reported to have inhibitory effects on lung cancer, liver cancer, breast cancer, colorectal cancer, and ovarian cancer, there are no reports on whether dendrobium oryzae has an inhibitory effect on ATC and the mechanism of its action. Summary of the Invention

[0005] The purpose of this invention is to provide a use of pyrifolia dendrobine against undifferentiated thyroid cancer. It has discovered a novel mechanism of action and a new indication for pyrifolia dendrobine—undifferentiated thyroid cancer, thus providing a new approach for the treatment of undifferentiated thyroid cancer.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] The application of a type of dendrobine in the preparation of a drug for treating undifferentiated thyroid cancer.

[0008] Dendrobium truncatum extract inhibits the growth of undifferentiated thyroid cancer cells in vivo.

[0009] Dendrobium spp. inhibits the progression of undifferentiated thyroid cancer by regulating spleen tyrosine kinase to promote M1 macrophage polarization.

[0010] A drug for treating undifferentiated thyroid cancer, comprising a therapeutically effective amount of dendrobine.

[0011] The use of spleen tyrosine kinase as a target for dendrobium oryzae in the treatment of undifferentiated thyroid carcinoma.

[0012] The applicant's research found that dendrobium moniliforme can significantly inhibit ATC proliferation and colony formation, induce apoptosis, and arrest the cell cycle at the G0 / G1 phase, suggesting that dendrobium moniliforme plays an important role in inhibiting the malignant phenotype of ATC.

[0013] Through in vivo model experiments, the inventors discovered that *Dendrobium arylatum* extract can significantly inhibit the growth of ATC tumors. Further flow cytometry experiments showed that administration of *Dendrobium arylatum* extract upregulated M1 macrophages, suggesting that *Dendrobium arylatum* extract may be a drug for ATC immunotherapy. The applicant, through in vitro macrophage models, found that *Dendrobium arylatum* extract can increase the number of CD86 macrophages and decrease the number of CD163 macrophages, suggesting that *Dendrobium arylatum* extract can promote M1 macrophage polarization.

[0014] The inventors further discovered through in vitro T-cell proliferation experiments that *Dendrobium aryrifolium* extract could not promote T-cell proliferation. Previous studies have shown that T-cell proliferation depends on macrophages; however, whether macrophages can affect the function of cytotoxic T cells through *Dendrobium aryrifolium* extract requires further investigation. Meanwhile, whether *Dendrobium aryrifolium* extract participates in T-cell immune responses remains unclear. PD-1 antibodies are the most commonly used immune checkpoint inhibitors (ICIs), but the efficacy of single-drug therapy is limited by frequent drug resistance, and their clinical efficacy in ATC is not ideal, with an objective response rate of only about 20%. Modulating the ATC microenvironment is a potential solution to improve the response rate of ICIs.

[0015] Through network pharmacology research, the inventors discovered that spleen tyrosine kinase (SYK) is an important target of dendrobium oryzae in its anti-ATC response. Since SYK participates in the polarization process of M1 macrophages, database analysis determined that SYK is mainly located in macrophages. The applicant further confirmed through molecular docking and kinetic experiments that SYK has a good binding effect with dendrobium oryzae, speculating that SYK may be a key target for dendrobium oryzae in regulating ATC. Further immunofluorescence experiments showed that SYK is co-expressed in macrophages, suggesting that SYK may be a key target for dendrobium oryzae in regulating M1 macrophage polarization and anti-ATC response. Based on the above hypothesis, we used PCR and Western blotting to detect the expression level of SYK in macrophages and to investigate the effect of Dendrobium aryrifolium on SYK protein expression in macrophages. By knocking out SYK in macrophages, we investigated the dependence of Dendrobium aryrifolium on M1 macrophage polarization. Furthermore, we used Western blotting to confirm the dose-response relationship between Dendrobium aryrifolium inducing or inhibiting SYK expression. By establishing an ATC tumor-macrophage co-culture model, we investigated the effect of Dendrobium aryrifolium on SYK expression in macrophages under this model. Finally, we confirmed the novel function of Dendrobium aryrifolium in regulating tumor-associated macrophages and elucidated its novel tumor biological function as an epigenetic regulator. Based on research reports from domestic and international peers and the research foundation of our research group, we propose a scientific hypothesis: Dendrobium moniliforme exerts an inhibitory effect on ATC by regulating SYK expression and promoting M1 macrophage polarization, thus providing a new target for ATC treatment.

[0016] The beneficial effects of this invention are:

[0017] This invention discovers a novel pathway of action and a new indication for arytenoid dendrobine—undifferentiated thyroid carcinoma, and for the first time confirms the objective existence of arytenoid dendrobine in the anti-tumor activity of undifferentiated thyroid carcinoma.

[0018] This invention can provide more reliable targeted therapy for the initial treatment of undifferentiated thyroid cancer, simplify the treatment process, play a significant role in the diagnosis and treatment of undifferentiated thyroid cancer, and provide new ideas for the early prevention and treatment of undifferentiated thyroid cancer in clinical practice. Attached Figure Description

[0019] Figure 1 CCK8 assay was used to detect the effects of a series of compounds on the proliferation of ATC cell line 8505C at 48 h. Data are expressed as mean ± standard deviation. * P <0.05,** P <0.01, *** P <0.001;

[0020] Figure 2 Effects of Dendrobium moniliforme on ATC cell proliferation and clonal variation: A. CCK8 assay to detect the effect of Dendrobium moniliforme on 8505C cell proliferation after 48 h; B. Plate colony assay to detect the effect of Dendrobium moniliforme on colony formation ability of 8505C cells. Data are expressed as mean ± standard deviation, * P <0.05,** P <0.01, *** P <0.001;

[0021] Figure 3 The effect of Dendrobium chrysogenum extract on cell apoptosis was detected by flow cytometry. Data are expressed as mean ± standard deviation. * P <0.05,** P <0.01, *** P <0.001;

[0022] Figure 4 The effect of Dendrobium chrysogenum extract on cell cycle determination by flow cytometry. Data are expressed as mean ± standard deviation, * P <0.05,** P <0.01, *** P <0.001;

[0023] Figure 5 Dendrobium spp. inhibits ATC tumor growth; A. Tumor growth curve; B. Mouse body weight curve; C. Gross tumor appearance; D. Tumor weight. Data are expressed as mean ± standard deviation, * P <0.05,** P <0.01, *** P <0.001;

[0024] Figure 6 Effects of Dendrobium officinale on M1 / M2, MDSC, CD4, CD8 and NK cell counts in tumor tissues and spleen tissues of MATC subcutaneous undifferentiated thyroid carcinoma mice (n=6); A1. M1 / M2 macrophage count in tumor tissue; A2-A4. MDSC cell count in tumor tissue; A5. CD4 cell count in tumor tissue; A6. CD8 cell count in tumor tissue; A7. NK cell count in tumor tissue;

[0025] B1. M1 / M2 macrophage count in spleen tissue; B2-B4. MDSC cell count in spleen tissue; B5. CD4 cell count in spleen tissue; B6. CD8 cell count in spleen tissue; B7. NK cell count in spleen tissue;

[0026] Figure 7 A dose-dependent effect of pyrenoidin on the polarization of M1 macrophages was observed. Flow cytometry analysis of the expression of M0 macrophage markers F4 / 80, M1 macrophage marker CD86, and M2 macrophage marker CD163 showed that pyrenoidin promoted M1 macrophage polarization in a dose-dependent manner.

[0027] Figure 8 Effects of Dendrobium oryzae extract on T cell proliferation; AB. Flow cytometry analysis of the effects of Dendrobium oryzae extract on T cell proliferation.

[0028] Figure 9 A. Key target diagram of Dendrobium arylatum anti-ATC; B. Network construction diagram of Dendrobium arylatum-gene target; C. Hithubs network diagram of Dendrobium arylatum anti-ATC target protein; D. Visualization of SYK expression in various cell types of thyroid cancer using the TISCH database; E. Analysis of the binding effect of Dendrobium arylatum and SYK using molecular docking binding kinetics experiments. The results show that KSYK forms two hydrogen bonds with amino acid residue THR-64 in Dendrobium arylatum and forms pi-cation conjugation with amino acid residue TYR-63 in Dendrobium arylatum.

[0029] Figure 10 Fluorescence image of SYK co-expression in ATC cells. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0031] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0032] This invention utilizes CCK8 and plate cloning experiments to discover that *Dendrobium arylatum* extract can significantly inhibit ATC proliferation and colony formation. Apoptosis experiments were used to explore the ability of *Dendrobium arylatum* extract to promote ATC cell apoptosis, and cell cycle experiments were used to explore its ability to arrest the ATC cell cycle. An in vivo ATC model was established to verify the anti-ATC efficacy of *Dendrobium arylatum* extract. Further, flow cytometry, in vitro macrophage models, and T cell proliferation experiments were used to discover that *Dendrobium arylatum* extract may be a drug for ATC immunotherapy. Finally, network pharmacology, molecular docking, and immunofluorescence experiments were conducted to find that *Dendrobium arylatum* extract inhibits ATC progression by regulating SYK to promote M1 macrophage polarization, providing a new targeted intervention strategy for ATC treatment.

[0033] Example 1:

[0034] method

[0035] 1. Screening of antitumor activity of dendrobine: ATC cell line 8505C in good growth condition was seeded into 96-well plates at a density of 3000 cells / mL, 100 μL per well, and cultured in an incubator (37 ℃, CO2 5%) for 24 h. The blank control group was cultured in DMEM medium. The experimental group contained a series of natural small molecule compounds (dendroalkaloids, dendrobine, dendrobine, diosgenin, (-)-syringin, typhainoside (corresponding to...)). Figure 1 The concentration gradients of the compounds (English names of the compounds) (0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0 μmol / L) were set up, with 5 replicates in each group. The cells were incubated in an incubator for 48 h. The effects of a series of compounds on the proliferation of ATC cell line 8505C were detected by CCK8 assay to confirm the activity of Dendrobium chrysogenum in the fight against undifferentiated thyroid cancer.

[0036] 2. Effects of Dendrobium moniliforme on the malignant phenotype of ATC cells

[0037] 2.1 Cell proliferation detection

[0038] ATC cell lines 8505C, CAL62, and mATC, all in good growth condition, were seeded into 96-well plates at a density of 3000 cells / mL (100 μL per well) and incubated at 37 °C, 5% CO2 for 24 h. The control group was cultured in DMEM medium. Experimental groups were prepared with a series of concentration gradients of dendrobine (0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0 μmol / L), with 5 replicates per group. Cells were incubated for 48 h, and the effect of dendrobine on ATC cell proliferation was detected using CCK8 assay. Plate colony assay was used to evaluate the plate colony ability of dendrobine on ATC cells.

[0039] 2.2 Apoptosis Detection

[0040] Healthy ATC cell lines 8505C, CAL62, and mATC were seeded into 6-well plates at a density of 10 × 10⁻⁶ cells / well. 4 Cells were cultured at a concentration of 2 mL / mL in each well for 24 h. A blank control group was cultured in culture medium. The experimental groups were prepared with concentration gradients of Dendrobium officinale extract at IC50 and 1 / 2 IC50, with three replicates per group. Cells were cultured for 48 h, and the supernatant was collected. Cells were then digested with trypsin (without EDTA), centrifuged at 3000 r / min, 4 ℃ for 5 min, the supernatant was discarded, and the cells were collected. Cells were washed twice with pre-chilled PBS (0.01M, pH 7.2-7.4), and resuspended in 500 μL Binding Buffer (Hangzhou Lianke Biotechnology Co., Ltd.). 5 μL Annexin V-FITC (Hangzhou Lianke Biotechnology Co., Ltd.) and 10 μL PI staining solution (Hangzhou Lianke Biotechnology Co., Ltd.) were added, mixed, and incubated at room temperature in the dark for 5 min. The cells were then placed on ice and analyzed by flow cytometry within 1 h.

[0041] 2.3 Cell cycle detection

[0042] Healthy ATC cell lines 8505C, CAL62, and mATC were seeded into 6-well plates at a density of 10 × 10⁻⁶ cells / well. 4Cells were cultured at a concentration of 2 mL / mL in each well for 24 h. The blank control group was cultured in culture medium. The experimental group was set with a concentration gradient of Dendrobium chrysogenum extract containing IC50 and 1 / 2 IC50. Each group had 3 replicates. Cells were cultured in an incubator for 48 h. The culture medium was discarded, and the cells were washed twice with pre-cooled PBS. After removing the PBS, the cells were digested with trypsin without EDTA and collected. The cells were centrifuged at 3000 r / min, 4 °C for 5 min, the supernatant was discarded, and the cells were collected. The cells were washed twice with pre-cooled PBS and resuspended in 0.5 mL of pre-cooled PBS to fully suspend the cells into single cells. The resuspended cells were then added to 1.2 mL of pre-cooled 99.7% anhydrous ethanol (final ethanol concentration 70%), and the mixture was pipetted to avoid cell aggregation. The cells were fixed at 4 °C for 2 h to overnight. After washing the cells with PBS, the cell pellet was collected again, and 100 μL of RNase A (Hangzhou Lianke Biotechnology Co., Ltd.) was added and the mixture was incubated at 37 ℃ for 30 min. Finally, 400 μL of PI was added and the cells were stained in the dark for 30 min. The cells were then analyzed by flow cytometry.

[0043] 2.4 Cell migration detection

[0044] Healthy ATC cell lines 8505C, CAL62, and mATC were seeded into 6-well plates at a density of 10 × 10⁻⁶ cells / well. 4 Cells were cultured at a density of 2 mL / well for 24 h in an incubator. A blank control group was cultured in culture medium. The experimental groups were set up with concentration gradients of Dendrobium chrysogenum extract containing IC50 and 1 / 2 IC50, with 3 replicates per group. Cells were cultured for 48 h, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. After removing the PBS, the cells were trypsinized and collected. The cells were centrifuged at 1000 r / min, 4 ℃, for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of culture medium to ensure complete single-cell resuscitation. Tumor cells were then seeded into the upper chamber of a Transwell at a density of 5 × 10⁶ cells / well. 4 Cells / mL, 200 μL per chamber, 700 μL of serum-free medium (DMEM) added to the lower chamber of the Transwell, 3 replicates per group, incubated in an incubator for 48 h, discard the medium from the upper and lower chambers of the Transwell, wash twice with pre-cooled PBS, remove the PBS, fix with paraformaldehyde overnight, discard the paraformaldehyde from the upper and lower chambers of the Transwell, stain with crystal violet for 15 min, wash twice with pre-cooled PBS, remove the PBS, use a cotton swab to gently swab away the remaining cells on the upper surface of the Transwell chamber, avoid puncturing the filter membrane, the stained cells obtained are the migrating cells, photographed under a microscope, and the effect of pyrogallol on ATC cell migration was analyzed.

[0045] 2.5 Cell invasion detection

[0046] Healthy ATC cell lines 8505C, CAL62, and mATC were seeded into 6-well plates at a density of 10 × 10⁻⁶ cells / well. 4 Cells were cultured at a concentration of 2 mL / well for 24 h in an incubator. The blank control group was cultured in culture medium. The experimental groups were set with concentration gradients of Dendrobium officinale solution containing IC50 and 1 / 2 IC50, with 3 replicates per group. The cells were cultured in an incubator for 48 h, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. After removing the PBS, the cells were digested with trypsin and collected. The cells were centrifuged at 1000 r / min, 4℃, for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of culture medium to ensure that the cells were fully suspended into single cells. Matrigel (Corning Comfort Technology, USA) was thawed overnight at 4℃ and prepared as a liquid. The Matrigel was diluted 10 times with culture medium. The pipette tip was inserted below the liquid surface and blown to ensure that it was mixed evenly and without air bubbles. 70 μL of Matrigel was evenly distributed on the upper surface of the chamber and incubated in an incubator for 30 min. Tumor cells were seeded into the upper chamber of a Transwell at a density of 8 × 10⁴ cells / mL, 200 μL per chamber. 700 μL of serum-free culture medium was added to the lower chamber of the Transwell. Three replicates were set up for each group. The cells were incubated for 48 h. The culture medium from both the upper and lower chambers of the Transwell was discarded. The cells were washed twice with pre-cooled PBS, the PBS was removed, and the cells were fixed overnight with paraformaldehyde. The paraformaldehyde in both the upper and lower chambers of the Transwell was discarded. The cells were stained with crystal violet for 15 min, washed twice with pre-cooled PBS, the PBS was removed, and the remaining cells on the upper surface of the Transwell chamber were gently removed with a cotton swab in a rotating motion to avoid puncturing the filter membrane. The resulting stained cells were identified as invasive cells. Microscopic images were taken to analyze the effect of Dendrobium officinale on ATC cell invasion.

[0047] 3. In vitro and in vivo studies on the anti-tumor effects of dendrobium officinale on tumor-associated macrophages.

[0048] 3.1 Therapeutic effect of Dendrobium arylatum extract on mATC undifferentiated thyroid carcinoma mice: Eighteen 6-week-old female C57BL / 6 mice weighing 17-20g were randomly divided into a blank control group, a low-dose Dendrobium arylatum extract group (50 mg / kg), and a high-dose Dendrobium arylatum extract group (100 mg / kg) after 7 days of acclimatization, with 6 mice in each group. mATC tumor cells were collected at a density of 1 × 10⁻⁶. 6 A cell suspension of 1 cell / mL was inoculated subcutaneously in the axilla of the right forelimb of mice. Weight changes were continuously monitored, and the mice were sacrificed after 4 weeks. Tumor diameters of different groups were compared.

[0049] 3.2 Regulation of macrophages, T cells, NK cells and MDSC cells in tumor tissues and spleen tissues of mice with mATC undifferentiated thyroid carcinoma by Dendrobium chrysogenum.

[0050] (1) Regulation of macrophages, T cells, NK cells and MDSC cells in tumor tissue of mice with mATC undifferentiated thyroid carcinoma by dendrobine

[0051] 3.2.1 Tumor Digestion: Weigh an appropriate amount of collagenase IV powder and dissolve it in serum-free culture medium (DMEM) to prepare a 1 mg / mL tumor tissue digestion solution. Cut the tumor tissue into 1 mm × 1 mm × 1 mm pieces and place them in a 50 mL centrifuge tube. Add 10 mL of the digestion solution to the centrifuge tube, tighten the cap, and label the tumor group and serial number on the cap and tube. Fix the centrifuge tube containing the tumor pieces and digestion solution in a water bath shaker. Adjust the instrument parameters to a shaking speed of 300 mpm and a temperature of 37 ℃. Digest in a water bath for about 1 hour until the tumor pieces can no longer be digested and the liquid in the centrifuge tube becomes turbid. The shaking time should not be too long, otherwise it will damage the cell state and be detrimental to subsequent flow cytometry detection. The tumor digestion solution was filtered through a 70 μM filter into a 15 mL centrifuge tube. The tubes were centrifuged at 1000 r / min, 4 ℃, for 5 min. The supernatant was discarded, and the tumor cells were resuspended in 3 mL of PBS via pipette. The cells were centrifuged again at 1000 r / min, 4 ℃, for 5 min, and washed twice with PBS. The Zombie Red™ Dye (BioLegend) was diluted 1000-fold with PBS. 400 μL of the diluted dye was added to each tube of tumor cells, and the cells were resuspended. The cells were stained on ice in the dark for 30 min. The tumor cells were then resuspended and divided into two equal volumes, which were transferred to 1.5 mL EP tubes. The 1.5 mL EP tubes were labeled with group numbers.

[0052] 3.2.2 Flow Cytometry Staining (Cell Membrane Staining): The cell membrane staining antibodies (BioLegend) required for the experiment are Anti-CD45, Anti-CD3, Anti-CD4, Anti-CD8, Anti-NK1.1, Anti-CD11b, Anti-F4 / 80, Anti-CD86, Anti-CD163, Anti-MHCII, Anti-CD1lc, Anti-Gr-1, Anti-LY6G, and Anti-LY6C. Add 1 μL of antibody + 200 μL of PBS to each tube of tumor cells, fully resuspend the cells, and ensure that the cell staining solution is in full contact with the antibody. Stain on ice in the dark for 30-40 min, centrifuge, discard the supernatant, add 1 mL of FACS washing buffer (BioLegend) to each tube to wash the cells, centrifuge at 5000 r / min, 4℃ for 5 min, discard the supernatant, resuspend the cells in 200 μL of FACS, and perform flow cytometry analysis. If not testing on the same day, add 200 μL of fixative (FOXP3 Perm Buffer 10x (BioLegend) diluted to 1x with PBS) and fix for 30 min (protect from light). Centrifuge, discard the supernatant, add 1 mL of FACS washing buffer to each tube to wash the cells, centrifuge at 5000 r / min, 4 ℃ for 5 min, discard the supernatant, resuspend the cells with 1 mL of FACS, and store at 4 ℃ protected from light for up to one week.

[0053] 3.2.3 Flow Cytometry Staining (Nuclear Staining): Cell membrane staining is performed in the same manner as described above. After cell membrane staining, fixation buffer (Fixation / Permeabilization Concentrate: Fixation / Permeabilization Diluent = 1:3 (BioLegend)) is used to perforate cells at room temperature in the dark for 15-30 min. Simultaneously, Permeabilization Bufer (10x) (BioLegend) is diluted to 1x with PBS. After perforation, the supernatant is discarded by centrifugation. Anti-FOXP3 staining buffer is prepared using the same method as the flow cytometry antibody preparation for cell membrane staining. The staining method is also the same. After staining, 1 mL of Permeabilization Bufer (1x) (BioLegend) is added to each tube, centrifuged at 5000 rpm at room temperature for 5 min, the supernatant is discarded, and 200 μL of Permeabilization Bufer (1x) is added to each tube for fixation. The cells are then analyzed using Flowio software.

[0054] (2) Regulation of macrophages, T cells, NK cells and MDSC cells in spleen tissue of mice with mATC undifferentiated thyroid cancer by dendrobine

[0055] 3.2.4 Extraction of cells from spleen tissue: Add 5 mL of PBS to a culture dish, place the spleen in the dish, and crush the spleen thoroughly with a syringe or pipette tip. Mix thoroughly with a pipette and filter through a 70 μM filter into a 15 mL centrifuge tube. Centrifuge at 1000 r / min, 4 ℃, for 5 min, discard the supernatant, and resuspend the tumor cells in 3 mL of PBS. Centrifuge at 1000 r / min, 4 ℃, for 5 min, discard the supernatant, add 3 mL of 1x erythrocyte lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), resuspend the cells, and incubate for 5 min to fully lyse the erythrocytes. Centrifuge at 1000 r / min, 4 ℃, for 5 min, discard the supernatant, and wash the cells twice with PBS. Resuspend the cells in 1 mL of PBS, divide the cells into two equal volumes, and transfer them to 1.5 mL EP tubes. Label the 1.5 mL EP tubes with group numbers.

[0056] 3.2.5 Flow cytometry staining (cell membrane staining): The method is the same as 3.2.2.

[0057] 3.2.6 Flow cytometry staining (nuclear staining): The method is the same as in 3.2.3.

[0058] 3.3 In vitro study of the regulation of tumor-associated macrophage polarization by dendrobium chrysogenum.

[0059] (1) Extraction of mouse bone marrow-derived macrophages (BMDM): C57BL / 6 mice were euthanized by cervical dislocation and immersed in 70% anhydrous ethanol for 1-2 min. Three small dishes were filled with an appropriate amount (5 mL) of PBS. Three paper towels were placed in the biosafety cabinet, and scissors, tweezers, and a blade were prepared. The mice were taken out and placed on the paper towels. The skin of the mouse's hind leg was cut open with scissors, and the skin was separated from the muscle with tweezers. The muscle was scraped clean along the tibia and femur of the hind leg with a blade (scraping vertically, not cutting) until the hip joint (there is a lot of muscle in the femur, which can be cut with scissors, but blood vessels should be avoided). (Operate on both legs together), cut the connection at the hip joint, remove the femur and tibia, wash once in pre-cooled sterile PBS, continue to remove muscle with a blade, and cut the connection at the femur and tibia, and the connection at the tibia and ankle, wash once in pre-cooled sterile PBS, place the femur and tibia in new pre-cooled sterile PBS, take a section of leg bone, cut the epiphyseal ends appropriately, use a 26 G syringe to draw 0.5-1 mL of PBS and insert it into the bone marrow cavity from one end (as close to the top as possible) to rinse the bone marrow. After the leg bone turns white, rinse the other end (the leg bone can be cut after it turns white to facilitate rinsing the remaining part), gently aspirate and disperse the bone marrow cells with a 1 mL pipette tip until there is no red flocculent matter, pass the evenly suspended cells through a 40 μM nylon sieve to remove cell clumps, transfer to a centrifuge tube, centrifuge at 1000 r / min, 4 ℃ for 5 min, discard the supernatant, and obtain mouse bone marrow-derived macrophages BMDM.

[0060] (2) Polarization of mouse bone marrow-derived macrophages BMDM: Transfer cells from centrifuge tubes to 6-well plates (it is recommended that bone marrow macrophages extracted from a normal-sized adult C57BL / 6 mouse (about 20 g) be seeded into 4 wells of a 6-well plate), add M-CSF (Wuhan Aibote Biotechnology Co., Ltd.) to a final concentration of 20 ng / mL, mix well and incubate for three days. After three days, wash the cells two to three times with preheated PBS (to wash away red blood cells), change the medium with DMEM medium (DMEM + 10% FBS + 1% penicillin antibody + 20 ng / mL M-CSF); continue to incubate for three days to obtain BMDM macrophages.

[0061] (3) Regulatory effect of Dendrobium arylatum on macrophage polarization in vitro: Macrophages were treated with Dendrobium arylatum and cultured in an incubator for 48 h. The expression of M0 macrophage marker F4 / 80, M1 macrophage marker CD86 and M2 macrophage marker CD163 was analyzed by flow cytometry to explore the regulatory effect of Dendrobium arylatum on macrophage polarization in vitro.

[0062] (4) Tumor-macrophage interaction in a cell co-culture model using Dendrobium oryzae extract: mATC tumor cells were seeded in the upper chamber of a Transwell, and M-CSF-induced BMDM macrophages were seeded in the lower chamber of a Transwell to construct a tumor-macrophage co-culture model. A Dendrobium oryzae extract solution containing Dendrobium oryzae extract was co-incubated with mATC tumor cells in the upper chamber for 48 h. After removing the upper chamber, the levels of F4 / 80, CD86, and CD163 in the BMDM macrophages in the lower chamber were measured by flow cytometry.

[0063] 4. In vivo and in vitro studies on the effects of dendrobium chrysogenum extract on blocking the immunosuppressive microenvironment and on the function of cytotoxic T cells.

[0064] 4.1 Blocking effect of Dendrobium arylatum on the immunosuppressive microenvironment: Macrophages were treated with Dendrobium arylatum and cultured in an incubator for 48 h. The culture supernatant of macrophages after Dendrobium arylatum treatment was collected, and the levels of IL-1β, IL-2, IL-6, IL-8, IL-10, TNF-α, IFN-γ, TGF-β, and GM-CSF were detected according to the ELISA kit (Wuhan Aibote Biotechnology Co., Ltd.). The effect of Dendrobium arylatum on macrophage cytokine secretion was investigated, and the blocking effect of Dendrobium arylatum on the immunosuppressive microenvironment was analyzed.

[0065] 4.2 Effects of Dendrobium moniliforme on the function of cytotoxic T cells in vitro

[0066] CD3 antibody was used to coat plates, which were then incubated overnight at 4 °C. Mouse-derived spleen T cells were extracted and stained with CFSE (BioLegend) in the dark. (1 μL (1000x) of CFSE can be used for 1 × 10⁻⁶ cells.) 7After staining T cells (20 min), wash twice with PBS and set aside. Use unstained T cells as a negative control by flow cytometry, adjusting parameters to a suitable size (FITC channel is sufficient). Then, use the stained T cells as a positive control by flow cytometry. Seed the remaining stained T cells into 96-well plates, adding different concentrations of dendrobium officinale and co-culturing with T cells according to different groups. After incubation at 37 ℃ for 72 h, collect the cells, centrifuge at 1000 r / min, 4 ℃ for 5 min, block the cells with 5% BSA solution, centrifuge at 1000 r / min, 4 ℃ for 5 min, collect the cells, and stain with CD3 flow cytometry antibody (BioLegend) (FITC cannot be used; it needs to be protected from CFSE light; PE / APC can be used) in the dark. After a few minutes, the cells were washed twice with PBS and then analyzed by flow cytometry. The CFSE status was observed in the CD3+ group to investigate the effect of Dendrobium arylatum on T cell proliferation. The same method was used to coat T cells with CD3 antibody, extract T cells, and perform CFSE staining. Macrophages treated with Dendrobium arylatum were co-cultured with T cells and incubated at 37 °C for 72 h. Cells were collected, stained, and analyzed by flow cytometry in the same manner as above to investigate changes in cytotoxic T cell function, in order to determine whether macrophages can affect cytotoxic T cell function through Dendrobium arylatum.

[0067] 4.3 Effect of Dendrobium tumefaciens extract on the immune checkpoint PD-1 immunotherapy in mATC undifferentiated thyroid carcinoma mice

[0068] Twenty-four 6-week-old female C57BL / 6 mice, weighing 17–20 g, were randomly divided into four groups after 7 days of acclimatization: a blank control group, a dendrobium officinale group (100 mg / kg), a PD1 group (2 mg / kg), and a combination group of dendrobium officinale (100 mg / kg) + PD1 (2 mg / kg). Six mice were used in each group. mATC tumor cells were introduced at a density of 1 × 10⁻⁶. 6 Cell suspensions of [number] cells / mL were subcutaneously inoculated into the right forelimb axilla of mice. Body weight changes were continuously monitored, and mice were sacrificed after 4 weeks. Tumor diameters were compared between different groups. Tumor tissue from the experimental mice was fixed, dehydrated, embedded, sectioned, and prepared into paraffin sections of mouse lung tissue. HE staining was performed on the blank control group, the *Dendrobium arylatum* group, the PD1 group, and the *Dendrobium arylatum* + PD1 combination group to analyze the effect of *Dendrobium arylatum* on the immune checkpoint PD-1 immunotherapy in mice with mATC undifferentiated thyroid carcinoma.

[0069] 4.4 Regulation of PD1 and CD152 cells in tumor tissues and spleen tissues of mice with mATC undifferentiated thyroid carcinoma: The number of PD1 and CD152 cells in tumor tissues and spleen tissues of mice in each group was detected by flow cytometry to explore the regulatory effect of dendrobium on PD1 and CD152 cells.

[0070] 5. Study on the effect of dendrobium tumefaciens extract on M1 macrophage protein expression through regulation of SYK.

[0071] 5.1 Network pharmacology study of dendrobium spice in anti-ATC activity: Potential targets of dendrobium spice were obtained using the online databases PharmMapper, TCMSP, SEASearchServer, and STITCH. Simultaneously, the GeneCards database was searched to identify ATC's host targets. The online program Draw VennyDiagram was used to obtain the intersection targets between dendrobium spice and ATC. A target protein interaction (PPI) network was constructed using the STRING database and Cytoscape 3.9.1 software, and key targets were screened. GO function and KEGG pathway enrichment analyses were performed on the targets.

[0072] 5.2 Molecular docking analysis of the optimal docking target of Dendrobium oryzae against ATC: Molecular docking of the core target and small molecules was performed using AutoDockVinav 1.2.0 software, and the binding energy and binding mode of Dendrobium oryzae to the target protein were analyzed. The docking results were visualized using PyMOLv2.5, and the optimal docking target of Dendrobium oryzae against ATC was finally determined.

[0073] 5.3 Effect of Dendrobium oryzae extract on SYK protein expression in macrophages: The effect of Dendrobium oryzae extract on SYK protein expression in macrophages was detected by PCR, immunofluorescence and Western blot.

[0074] 6. Study on the mechanism of action of dendrobium tumefaciens in regulating SYK expression, promoting M1 macrophage polarization, and fighting undifferentiated thyroid carcinoma.

[0075] 6.1 Dictamnus oryzae-based synergistic effect on M1 macrophage polarization: SYK was knocked out in macrophages, and the knockdown efficiency was assessed by transfecting macrophages with shRNA. After SYK knockdown, macrophages were treated with synergistic effect, and changes in macrophage function were examined to clarify that synergistic effect on M1 macrophage polarization is SYK-dependent.

[0076] 6.2 Dose-response relationship of dendrobium oryzae inducing or inhibiting SYK expression in macrophages: By administering different concentrations of dendrobium oryzae to macrophages at different time points, Western blot was used to examine changes in SYK expression in macrophages, confirming the dose-response relationship between dendrobium oryzae inducing or inhibiting SYK expression.

[0077] 6.3 Induction or Inhibition of SYK Expression in Macrophages by Dendrobium officinale in an ATC Tumor-Macrophage Co-culture Model: mATC tumor cells were seeded in the upper chamber of a Transwell, and MCSF-induced BMDM macrophages were seeded in the lower chamber of a Transwell to construct a tumor-macrophage co-culture model. Tumor cells were treated with different concentrations of Dendrobium officinale at different time points, and co-incubated for 48 h. Western blot analysis was then used to examine changes in SYK expression in macrophages.

[0078] Example 2: Results Analysis

[0079] 1. Dendrobium officinale inhibits ATC cell activity.

[0080] CCK8 assays were performed to detect the effects of a series of compounds on the proliferation of the ATC cell line 8505C. The CCK8 results showed that dendrobine had a strong inhibitory effect on ATC cells. Figure 1 ).

[0081] 2. Effects of Dendrobium chrysogenum extract on tumor cell proliferation and clonal variation.

[0082] To investigate the effect of cypermethrin on tumor cell proliferation, the effect of cypermethrin on the proliferation of ATC cell line 8505C for 48 h was detected using CCK8 assay. Plate colony assay was used to assess the plate colony-forming ability of cypermethrin on ATC cell line 8505C. CCK8 results showed that cypermethrin could inhibit the proliferation of ATC cell line 8505C (…). Figure 2 A) Cell proliferation. Further plate colony experiments showed that dendrobine significantly inhibited ATC cell line 8505C ( Figure 2 B) Cell plate cloning ability.

[0083] 3. Effects of Dendrobium nobile extract on apoptosis

[0084] Flow cytometry apoptosis assays showed that dendrobine significantly induced apoptosis in ATC cells. Figure 3 ).

[0085] 4. Dendrobium oryzae extract arrests the ATC cell cycle.

[0086] The effect of dendrobium oryzae extract on the cell cycle of ATC cells was detected by flow cytometry. The results showed that dendrobium oryzae extract could cause cell cycle arrest at 8505C in ATC cells and reduce the proportion of cells in the G0 / G1 phase. Figure 4 ).

[0087] 5. The inhibitory effect of Dendrobium chrysogenum extract on ATC tumor growth.

[0088] In C57BL / 6 female mouse xenograft experiments, dendrobium officinale significantly inhibited ATC cell tumor growth, while mouse body weight showed no significant change. Figure 5 AB). After reaching the experimental endpoint, mice were sacrificed and ectopic tumors were removed. It was found that the tumor volume and weight were significantly reduced in the group treated with Dendrobium officinale extract (100 mg / Kg). Figure 5 CD).

[0089] 6. Regulation of macrophages, MDSC cells, T cells, and NK cells in tumor tissues and spleen tissues of MATC undifferentiated thyroid carcinoma mice.

[0090] Flow cytometry analysis was performed to analyze the regulatory effects of Dendrobium chrysogenum extract on macrophages, T cells, NK cells, and MDSC cells in tumor tissues and spleen tissues of MATC undifferentiated thyroid carcinoma mice. Flow cytometry results from tumor tissues showed that Dendrobium chrysogenum extract upregulated M1 macrophages, and the increase in M1 macrophages in the Dendrobium chrysogenum extract group was statistically significantly different from that in the control group. Figure 6 -A1, P <0.05); Dendrobium officinale administration downregulated M2 macrophages, and the reduction effect of Dendrobium officinale on M2 macrophages was statistically different in the Dendrobium officinale group compared with the control group ( Figure 6 -A1, P <0.05); Dendrobium officinale administration upregulated CD8 cells, and the effect of Dendrobium officinale on increasing CD8 cells was statistically different in the group compared with the control group. Figure 6 -A6, P <0.001); Flow cytometry results of spleen tissue showed that administration of Dendrobium chrysogenum upregulated M1 macrophages, and the effect of Dendrobium chrysogenum on increasing M1 macrophages was statistically different in the Dendrobium chrysogenum group compared with the control group. Figure 6 -B1, P <0.05); Dendrobium officinale administration downregulated M2 macrophages, and the reduction effect of Dendrobium officinale on M2 macrophages was statistically different in the Dendrobium officinale group compared with the control group ( Figure 6 -B1, P <0.05); Dendrobium officinale administration upregulated MDSC cells, and the effect of Dendrobium officinale on increasing MDSC cells was statistically significant compared with the control group. Figure 6 -B2, P<0.01); Dendrobium officinale administration upregulated M-MDSC cells, and the effect of Dendrobium officinale on increasing M-MDSC cells was statistically different in the Dendrobium officinale group compared with the control group. Figure 6 -B3, P <0.001); Dendrobium officinale administration upregulated PMN-MDSC cells, and the effect of Dendrobium officinale on increasing PMN-MDSC cells was statistically significant compared with the control group. Figure 6 -B4, P <0.05).

[0091] 7. The regulatory effect of dendrobine on macrophage polarization

[0092] To investigate the regulatory effect of dendrobium oryzae extract on macrophage polarization in vitro, mouse bone marrow-derived macrophages (BMDM) were treated with dendrobium oryzae extract. The expression of M0 macrophage marker F4 / 80, M1 macrophage marker CD86, and M2 macrophage marker CD163 was analyzed by flow cytometry. The results showed that dendrobium oryzae extract promoted high expression of M1 macrophage marker CD86 and inhibited the expression of M2 macrophage marker CD163. Figure 7 AB).

[0093] 8. Effects of Dendrobium moniliforme on T cell proliferation

[0094] To investigate the effect of Dendrobium oryzae extract on T cell proliferation in vitro, a Dendrobium oryzae extract-T cell co-culture model was used to examine the effect of Dendrobium oryzae extract on T cell proliferation. Figure 8 (AB). Flow cytometry results showed that dendrobine from the chebula did not promote T cell proliferation.

[0095] 9. Investigating the key targets of dendrobium tumefaciens in regulating anti-ATC activity in tumor-associated macrophages based on network pharmacology, molecular docking, and kinetic experiments.

[0096] Network pharmacology was used to preliminarily determine that dendrobium oryzae extract can exert its anti-ATC effect through multiple targets and pathways in the host. The results showed that dendrobium oryzae extract has 57 potential anti-ATC targets. Figure 9 AB), HSP90AA1, ESR1, ERBB2, and MMP9 are potential core targets for dendrobium tumefaciens to exert its anti-ATC effect. Figure 9 C) Using the TISCH database, visualizations of SYK expression were created in various cell types of thyroid cancer, potentially indicating that SYK is an important target of dendrobium oryzae in regulating macrophage M1 polarization against ATC. Figure 9D) The binding effect of KSYK on Dendrobium oryzae and SYK was analyzed using molecular docking binding kinetics experiments. The results showed that KSYK forms two hydrogen bonds with amino acid residue THR-64 in Dendrobium oryzae and forms a pi-cation conjugation with amino acid residue TYR-63 in Dendrobium oryzae. Figure 9 E).

[0097] 10. Immunofluorescence assays validated that SYK is a key target of dendrobium oryzae in regulating anti-ATC activity in tumor-associated macrophages.

[0098] Immunofluorescence assays were used to verify whether SYK was co-expressed on ATC cells. The results showed that SYK was co-expressed on ATC cells. Figure 10 ).

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. The application of a type of dendrobine in the preparation of an immunotherapeutic drug for undifferentiated thyroid cancer.

2. The application according to claim 1, characterized in that, Dendrobium truncatum extract inhibits the growth of undifferentiated thyroid cancer cells in vivo.

3. The application according to claim 1, characterized in that, Dendrobium spp. inhibits the progression of undifferentiated thyroid cancer by regulating spleen tyrosine kinase to promote M1 macrophage polarization.

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