Use of cypripetin for resisting undifferentiated thyroid cancer

Abstract

The invention discloses an application of cypripetin in resisting undifferentiated thyroid cancer, the cypripetin resists growth of undifferentiated thyroid cancer cells in vivo, and the cypripetin promotes polarization of M1 macrophages to inhibit progression of the undifferentiated thyroid cancer by regulating spleen tyrosine kinase. According to the invention, a brand-new action way and a new indication, namely the undifferentiated thyroid cancer, of the cypripetin are found, and a new way is provided for treating the undifferentiated thyroid cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to use of dendrobium arytenoides in treating anaplastic thyroid cancer. Background Art

[0002] Thyroid cancer is the most common endocrine malignancy, with an increasing incidence rate. Anaplastic thyroid cancer (ATC) is the most malignant type of thyroid cancer, often accompanied by extrathyroidal invasion or metastasis. Its incidence accounts for approximately 2% of all thyroid cancers, but its mortality rate accounts for 50% of thyroid cancer-related deaths. Currently, there is no effective treatment. Therefore, identifying the mechanisms of occurrence and progression of ATC and potential therapeutic targets remains a hot topic and a challenge.

[0003] Exploring targets for tumor therapy is a topic of concern for scholars both domestically and internationally. Tumor-associated macrophages (TAMs), the most numerous infiltrating immune cells in the tumor microenvironment (TME), play a crucial role in the development and progression of various malignant tumors and are considered an important target for cancer treatment. Currently, there are three main anti-tumor treatment strategies targeting TAMs: clearing TAMs, inhibiting TAM recruitment, and regulating TAM polarization. Regulating TAM polarization, such as the natural product 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 numbers in ATC cell lines, but the regulatory mechanism remains unclear and requires further investigation.

[0004] Targeted regulation of TAMs has emerged as a new strategy for cancer immunotherapy. Recent studies have shown that a growing number of natural products can act as immunomodulators and play a significant role in regulating TAM polarization. Therefore, identifying new natural product agents that can modulate TAMs could fill the gap in the availability of natural medicines for cancer immunotherapy and provide more options for clinical cancer treatment. Dendrobium is a perennial evergreen herbaceous plant of the genus Dendrobium in the Orchidaceae family. It is primarily distributed in Australia, Europe, Asia, and in Southwest and South China provinces in China. It is an important ingredient in traditional Chinese medicine formulas for adjuvant cancer treatment. Moscatilin, a hydroxybenzoic acid compound extracted from the rhizome of Dendrobium, is a key active ingredient in Dendrobium plants of the Orchidaceae family and exhibits significant antitumor, antioxidant, anti-inflammatory, and neuroprotective activities. In recent years, its anti-tumor effects have garnered significant attention. Current research suggests that the anti-tumor effects of moscatilin are primarily achieved through the regulation of tumor cells. Although it has been reported that arytenoidin has an inhibitory effect on lung cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, etc., there has been no report on whether arytenoidin has an inhibitory effect on ATC and its mechanism of action. Summary of the Invention

[0005] The purpose of the present invention is to provide a use of arytenoid dendrobium in treating anaplastic thyroid cancer, and to discover a new action pathway and a new indication of arytenoid dendrobium - anaplastic thyroid cancer, thereby providing a new approach for the treatment of anaplastic thyroid cancer.

[0006] The technical solution adopted by the present invention to solve its technical problem is: Application of arytenoid dendrobium in the preparation of medicines for treating anaplastic thyroid cancer.

[0007] Dendrobium arytenoides inhibits the growth of anaplastic thyroid cancer cells in vivo.

[0008] Dendrobium arytenoids inhibit the progression of anaplastic thyroid cancer by promoting M1 macrophage polarization through regulating spleen tyrosine kinase.

[0009] A drug for treating anaplastic thyroid cancer, comprising a therapeutically effective amount of arytenoidin.

[0010] Use of spleen tyrosine kinase as a target for arytenoid dendrobium officinale against anaplastic thyroid cancer.

[0011] The applicant's research found that arytenoid dendrobium can significantly inhibit ATC proliferation and clone formation, induce cell apoptosis, and arrest the cell cycle in the G0 / G1 phase, suggesting that arytenoid plays an important role in inhibiting the malignant phenotype of ATC.

[0012] Through in vivo model experiments, the inventors discovered that arytenoid dendrobium can significantly inhibit ATC tumor growth. Further flow cytometry experiments showed that arytenoid administration upregulated M1 macrophages, suggesting that arytenoid may be a drug for ATC immunotherapy. Using an in vitro macrophage model, the applicants found that arytenoid can increase the number of CD86 macrophages and reduce the number of CD163 macrophages, suggesting that arytenoid can promote M1 macrophage polarization.

[0013] The inventors further discovered through in vitro T cell proliferation experiments that dendrobium arytenoids cannot 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 arytenoids requires further study. At the same time, whether dendrobium arytenoids participate in T cell immune responses is not yet clear. PD-1 antibodies are the most commonly used immune checkpoint inhibitors (ICIs). The effectiveness of single treatment is limited by frequent drug resistance. The 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.

[0014] Through network pharmacology studies, the inventors discovered that spleen tyrosine kinase (SYK) is a key target for arytenoid dendrobium in its anti-ATC activity. Since SYK is involved in the polarization of M1 macrophages, database analysis confirmed that SYK is primarily localized in macrophages. The applicants further determined through molecular docking and kinetic experiments that SYK binds well to arytenoid dendrobium, suggesting that SYK may be a key target for arytenoid dendrobium in regulating ATC. Further immunofluorescence experiments demonstrated that SYK is co-expressed in macrophages, suggesting that SYK may be a key target for arytenoid dendrobium in regulating M1 macrophage polarization and anti-ATC activity. Based on the above assumptions, we used PCR and Western Blot techniques to detect the expression level of SYK in macrophages, and investigated the effect of arytenoids on SYK protein expression in macrophages; by knocking out SYK in macrophages, we investigated the dependence of arytenoids on SYK in promoting M1 macrophage polarization; further confirmed the dose-effect relationship of arytenoids in inducing or inhibiting SYK expression by Western blot; by establishing an ATC tumor-macrophage co-culture model, we investigated the effect of arytenoids on macrophage expression of SYK in the ATC tumor-macrophage co-culture model, and finally confirmed the new function of arytenoids in regulating tumor-associated macrophages, clarifying the novel tumor biological function of arytenoids as an epigenetic regulatory factor. Combining the research reports of domestic and foreign peers with the research foundation of the research team, a scientific hypothesis was proposed: Dendrobium arytenoids can inhibit ATC by regulating SYK expression and promoting M1 macrophage polarization, providing a new target for the treatment of ATC.

[0015] The beneficial effects of the present invention are: The present invention discovered a completely new pathway of action of arytenoid dendrobium and a new indication - anaplastic thyroid cancer, and confirmed for the first time the objective existence of arytenoid dendrobium in anti-tumor effects in anaplastic thyroid cancer.

[0016] The present invention can provide more reliable targeted therapy for the initial treatment of anaplastic thyroid cancer, simplify the treatment process, play a significant role in the diagnosis and treatment of anaplastic thyroid cancer, and provide new ideas for the early clinical prevention and treatment of anaplastic thyroid cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 CCK8 assay was used to detect the effects of a series of compounds on the proliferation of ATC cell line 8505C for 48 h. The data are expressed as mean ± standard deviation. P <0.05,** P <0.01,*** P <0.001; Figure 2 Effects of dendrobium arytenoids on ATC cell proliferation and cloning. A. CCK8 assay for the effect of dendrobium arytenoids on 8505C cell proliferation after 48 h; B. Plate cloning assay for the effect of dendrobium arytenoids on the cloning ability of 8505C cells. Data are expressed as mean ± SD. * P <0.05,** P <0.01,*** P <0.001; Figure 3 Flow cytometry apoptosis detection of the effect of Dendrobium arytenoides on cell apoptosis. The data are expressed as mean ± standard deviation. P <0.05,** P <0.01,*** P <0.001; Figure 4 Flow cytometry was used to detect the effect of Dendrobium arytenoides on the cell cycle. Data are expressed as mean ± standard deviation, * P <0.05,** P <0.01,*** P <0.001; Figure 5 Dendrobium arytenoides inhibits ATC tumor growth; A. Tumor growth curve; B. Mouse weight curve; C. Tumor gross appearance; D. Tumor weight. Data are expressed as mean ± SD, * P <0.05,** P <0.01,*** P <0.001; Figure 6Effects of dendrobium arytenoids on the counts of M1 / M2, MDSC, CD4, CD8, and NK cells in tumor tissue and spleen tissue of mice bearing subcutaneous anaplastic thyroid cancer (MATC) (n=6); A1. M1 / M2 macrophage counts in tumor tissue; A2-A4. MDSC counts in tumor tissue; A5. CD4 cell counts in tumor tissue; A6. CD8 cell counts in tumor tissue; A7. NK cell counts in tumor tissue; B1. M1 / M2 macrophage counts in spleen tissue; B2-B4. MDSC counts in spleen tissue; B5. CD4 cell counts in spleen tissue; B6. CD8 cell counts in spleen tissue; B7. NK cell counts in spleen tissue; Figure 7 Dendrobium arytenoids promote the polarization of M1 macrophages in a dose-dependent manner. Figure AB. Flow cytometry analysis of the expression of M0 macrophage markers F4 / 80, M1 macrophage markers CD86, and M2 macrophage markers CD163 showed that Dendrobium arytenoids promote the polarization of M1 macrophages in a dose-dependent manner. Figure 8 Effect of Dendrobium arytenoids on T cell proliferation; AB. Flow cytometry analysis of the effect of Dendrobium arytenoids on T cell proliferation; Figure 9 Key targets of arytenoid dendrobium for anti-ATC activity; A. Venn diagram of arytenoid dendrobium for anti-ATC activity; B. Diagram of the arytenoid dendrobium-gene target network; C. Hithubs network diagram of arytenoid dendrobium for anti-ATC activity; D. Visualization of SYK expression in various thyroid cancer cell types using the TISCH database; E. Molecular docking and binding kinetics analysis of the binding effect of arytenoid dendrobium and SYK. The results showed that KSYK formed two hydrogen bonds with amino acid residue THR-64 in arytenoid dendrobium and formed a pi-cation conjugation with amino acid residue TYR-63 in arytenoid dendrobium. Figure 10 Fluorescence image of SYK co-expression in ATC cells. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below through specific embodiments.

[0019] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0020] The present invention uses CCK8 and plate cloning experiments to find that arytenoid dendrobium can significantly inhibit ATC proliferation and clone formation, explores the ability of arytenoid to promote ATC cell apoptosis through cell apoptosis experiments, explores the ability of arytenoid to block the ATC cell cycle through cell cycle experiments, verifies the efficacy of arytenoid against ATC by establishing an in vivo ATC model, further finds that arytenoid may be a drug for ATC immunotherapy through flow cytometry experiments, in vitro macrophage models and T cell proliferation experiments, and finally finds through network pharmacology, molecular docking and immunofluorescence experiments that arytenoid promotes M1 macrophage polarization to inhibit ATC progression by regulating SYK, providing a new targeted intervention strategy for the treatment of ATC.

[0021] Example 1: method 1. Screening of anti-tumor activity of dendrobium styracifolium: ATC cell line 8505C with good growth status was taken and inoculated into 96-well plates at a density of 3000 cells / mL, with 100 μL per well. The cells were cultured in an incubator (37°C, CO25%, the same below) for 24 h. The blank control group was cultured with culture medium (DMEM medium, the same below). The experimental group was set up with a series of natural small molecule compounds (dendrobeline, dendrobium phenol, dendrobium styracifolium, yam Ш, (-)-syringaresinol, typhaneuroside (corresponding to Figure 1 The cells were cultured 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 anti-anaplastic thyroid cancer activity of Dendrobium arytenoides.

[0022] 2. Effect of Dendrobium arytenoidin on the malignant phenotype of ATC cells 2.1 Cell proliferation assay Well-growing ATC cell lines 8505C, CAL62, and mATC were seeded into 96-well plates at a density of 3000 cells / mL, with 100 μL per well. The plates were cultured in an incubator (37°C, 5% CO2) for 24 hours. A blank control group was cultured with DMEM medium. Experimental groups were treated with a series of concentration gradients of dendrobium arytenoidin (0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, and 40.0 μmol / L), with five replicates per group. The plates were incubated for 48 hours. The effect of dendrobium arytenoidin on ATC cell proliferation was assessed by CCK8 assay. The cloning ability of dendrobium arytenoidin on ATC cells was assessed by plate cloning.

[0023] 2.2 Cell apoptosis detection ATC cell lines 8505C, CAL62, and mATC that are in good growth condition were seeded into 6-well plates at a density of 10 × 10 4 Cells were cultured in an incubator for 24 h. A blank control group was incubated with culture medium. Experimental groups were treated with a gradient of concentrations of a Dendrobium arytenoidin solution at the IC50 and 1 / 2 IC50 concentrations, with triplicate wells per group. The cells were cultured in an incubator for 48 h. The supernatant was collected, and the cells were then digested with EDTA-free trypsin and harvested. The cells were centrifuged at 3000 rpm at 4°C for 5 min, the supernatant discarded, and the cells were washed twice with pre-chilled PBS (0.01 M, pH 7.2-7.4). The cells were 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 stain (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.

[0024] 2.3 Cell cycle detection ATC cell lines 8505C, CAL62, and mATC that are in good growth condition were seeded into 6-well plates at a density of 10 × 10 4 Cells were cultured in an incubator at 400 nm 400 nm saturated fat content (500 nm) at 37°C for 24 h, 2 mL per well, and cultured in the incubator for 24 h. The blank control group was cultured with culture medium. The experimental group was set with a concentration gradient of IC50 and 1 / 2 IC50 containing the drug solution of Dendrobium arytenoids. Three replicate wells were set for each group and cultured in the incubator for 48 h. The culture medium was discarded, the cells were washed twice with pre-cooled PBS, the PBS was removed, and the cells were collected by digestion with EDTA-free trypsin. The cells were centrifuged at 3000 r / min and 4°C for 5 min, the supernatant was discarded, the cells were collected, washed twice with pre-cooled PBS, and the cell pellet was resuspended with 0.5 mL pre-cooled PBS to fully suspend the cells into single cells. The resuspended cells were then added with 1.2 mL pre-cooled 99.7% anhydrous ethanol (final ethanol concentration 70%), mixed by pipetting to avoid cell agglomeration, and 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. The cells were incubated in a 37°C water bath for 30 min. Finally, 400 μL of PI was added and the cells were stained in the dark for 30 min. Flow cytometry was then performed.

[0025] 2.4 Cell migration assay ATC cell lines 8505C, CAL62, and mATC that are in good growth condition were seeded into 6-well plates at a density of 10 × 104 The cells were cultured in an incubator for 24 h. The blank control group was cultured with culture medium. The experimental group was cultured with a concentration gradient of the arytenoid solution at IC50 and 1 / 2 IC50. Three replicate wells were set in each group and cultured in an incubator for 48 h. The culture medium was discarded and the cells were washed twice with pre-cooled PBS. The PBS was removed and the cells were collected by trypsin digestion. The cells were centrifuged at 1000 r / min and 4°C for 5 min. The supernatant was discarded and the cells were resuspended in 1 mL of culture medium to fully suspend the cells into single cells. The tumor cells were inoculated into the upper chamber of the Transwell to a density of 5 × 10 4 Cells were plated at 400 μL / mL, 200 μL per chamber, and 700 μL serum-free medium (DMEM medium) was added to the lower chamber of the Transwell. Three replicate wells were set up for each group and the cells were cultured in an incubator for 48 h. The culture medium in the upper and lower chambers of the Transwell were discarded, and the cells were washed twice with pre-cooled PBS. The PBS was removed and the cells were fixed with paraformaldehyde overnight. The paraformaldehyde in the upper and lower chambers of the Transwell were discarded, and the cells were stained with crystal violet for 15 min. The cells were washed twice with pre-cooled PBS, and the PBS was removed. The remaining cells on the upper surface of the Transwell chamber were removed with a cotton swab. The movements were gentle to avoid puncturing the filter membrane. The stained cells obtained were migrating cells. The cells were photographed under a microscope to analyze the effect of arytenoidin on ATC cell migration.

[0026] 2.5 Cell invasion assay ATC cell lines 8505C, CAL62, and mATC that are in good growth condition were seeded into 6-well plates at a density of 10 × 10 4Cells were cultured in an incubator for 24 hours. A blank control group was treated with culture medium. Experimental groups were treated with a concentration gradient of dendrobium arytenoidin solution at the IC50 and 1 / 2 IC50 concentrations. Three replicates were set up in each group and cultured in an incubator for 48 hours. The culture medium was discarded, and the cells were washed twice with pre-chilled PBS. The PBS was removed, and the cells were harvested by trypsinization and centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of culture medium until they were fully suspended as single cells. Matrigel (Corning Comfort Technologies, USA) was refrigerated at 4°C overnight and thawed to a liquid state for later use. Matrigel was diluted 10-fold with culture medium. Pipettes were inserted below the liquid surface to ensure uniform mixing and the absence of bubbles. 70 μL of Matrigel was evenly distributed on the upper chamber surface of the chamber and allowed to stand in an incubator for 30 minutes. Tumor cells were inoculated into the upper chamber of Transwell at a density of 8 × 104 cells / mL, with 200 uL per chamber. 700 uL serum-free culture medium was added to the lower chamber of Transwell. Three replicates were set up for each group and the cells were cultured in an incubator for 48 h. The culture medium in the upper and lower chambers of Transwell were discarded, and the cells were washed twice with pre-cooled PBS. The PBS was removed and the cells were fixed with paraformaldehyde overnight. The paraformaldehyde in the upper and lower chambers of Transwell were discarded, and the cells were stained with crystal violet for 15 min. The cells were washed twice with pre-cooled PBS, and the PBS was removed. The remaining cells on the upper surface of the Transwell chamber were removed with a cotton swab, and the movements were gentle to avoid puncturing the filter membrane. The stained cells were identified as invasive cells. The cells were photographed under a microscope to analyze the effect of arytenoidin on the invasion of ATC cells.

[0027] 3. In vitro and in vivo studies on the anti-tumor effects of Dendrobium arytenoides on tumor-associated macrophages 3.1 Therapeutic Effect of Dendrobium arytenoids on mATC Undifferentiated Thyroid Cancer Mice: Eighteen 6-week-old C57BL / 6 female mice weighing 17-20 g were randomly divided into a blank control group, a low-dose Dendrobium arytenoids group (50 mg / kg), and a high-dose Dendrobium arytenoids group (100 mg / kg) after adaptive feeding for 7 days, with 6 mice in each group. mATC tumor cells were cultured at a density of 1 × 10 6 The cell suspension of 100 cells / mL was inoculated subcutaneously in the axilla of the right forelimb of mice. The body weight changes were continuously monitored. The mice were killed after 4 weeks and the tumor diameters of different groups were compared.

[0028] 3.2 Regulation of Dendrobium arytenoids on macrophages, T cells, NK cells, and MDSCs in tumor tissues and spleen tissues of mATC anaplastic thyroid cancer mice (1) Regulation of Dendrobium arytenoids on macrophages, T cells, NK cells and MDSC cells in tumor tissues of mATC undifferentiated thyroid cancer mice 3.2.1 Tumor Digestion: Weigh an appropriate amount of collagenase IV powder and dissolve it in serum-free 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. Pipette 10 mL of digestion solution into the tube, tighten the cap, and label the tube with the tumor group and serial number. Place the tube containing the tumor and digestion solution in a waterbath shaker. Adjust the instrument parameters to a shaking speed of 300 mpm and a temperature of 37°C. Shake and digest in a waterbath for approximately 1 hour until the tumor is no longer digested and the liquid in the tube becomes turbid. Do not shake for too long, as this will damage the cells and hinder subsequent flow cytometry analysis. Filter the tumor digestion solution through a 70 μM filter into a 15 mL centrifuge tube and centrifuge at 1000 rpm at 4°C for 5 min. Discard the supernatant and pipette 3 mL of PBS to resuspend the tumor cells. Centrifuge at 1000 rpm at 4°C for 5 min. Wash the tumor cells twice with PBS. Dilute the anti-viability dye Zombie Red™ Dye (BioLegend) 1000-fold with PBS. Add 400 μL of the diluted anti-viability dye to each tube of tumor cells and resuspend the cells. Stain on ice in the dark for 30 min. Resuspend the cells and divide the tumor cells into two equal groups. Transfer the cells to 1.5 mL EP tubes. Label the tubes with the group numbers.

[0029] 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-CDl1b, Anti-F4 / 80, Anti-CD86, Anti-CD163, Anti-MHCII, Anti-CD1lc, Anti-Gr-1, Anti-LY6G, and Anti-LY6C. 1 μL of antibody plus 200 μL of PBS was added to each tube of tumor cells. The cells were fully resuspended to allow for full contact between the cell staining solution and the antibody. The cells were stained on ice in the dark for 30-40 min. The cells were centrifuged and the supernatant was discarded. 1 mL of FACS wash buffer (BioLegend) was added to each tube to wash the cells. The cells were centrifuged at 5000 rpm at 4°C for 5 min. The supernatant was discarded and the cells were resuspended in 200 μL of FACS buffer for analysis. If the test is not being performed on the same day, add 200 μL of fixative (FOXP3 Perm Buffer 10x (BioLegend) diluted to 1x with PBS) and fix for 30 min (keep away from light). Centrifuge and discard the supernatant. Add 1 mL of FACS wash buffer to each tube to wash the cells. Centrifuge at 5000 rpm at 4°C for 5 min. Remove the supernatant and resuspend the cells in 1 mL of FACS buffer. Store at 4°C in the dark for up to one week.

[0030] 3.2.3 Flow Cytometry Staining (Nuclear Staining): Cell membrane staining is performed in the same manner as described above. After membrane staining, use fixative (Fixation / Permeabilization Concentrate: Fixation / Permeabilization Diluent = 1:3 (BioLegend)) to perforate the cells in the dark for 15-30 minutes at room temperature. Simultaneously, dilute Permeabilization Bufer (10x) (BioLegend) to 1x with PBS. After perforation, centrifuge and discard the supernatant. Prepare flow cytometry Anti-FOXP3 staining solution using the same preparation and staining methods as for cell membrane staining. After staining, add 1 mL of Permeabilization Bufer (1x) (BioLegend) to each tube and centrifuge at 5000 rpm for 5 minutes at room temperature. Discard the supernatant and fix the cells with 200 μL of Permeabilization Bufer (1x). Analyze the cells on a flow cytometer and analyze the results using Flowio software.

[0031] (2) Regulation of Dendrobium arytenoids on macrophages, T cells, NK cells and MDSC cells in the spleen tissue of mATC undifferentiated thyroid cancer mice 3.2.4 Extracting Spleen Cells: Add 5 mL of PBS to a culture dish, place the spleen in the dish, crush the spleen thoroughly with a syringe or pipette, and pipette thoroughly to evenly distribute the spleen. Filter the mixture through a 70 μM filter into a 15 mL centrifuge tube, centrifuge at 1000 rpm at 4°C for 5 min, discard the supernatant, pipette 3 mL of PBS to resuspend the tumor cells, centrifuge at 1000 rpm at 4°C for 5 min, discard the supernatant, add 3 mL of 1x Red Blood Cell Lysis Buffer (Beijing Solebao Technology Co., Ltd.), resuspend the cells, let stand for 5 min to fully lyse the red blood cells, centrifuge at 1000 rpm at 4°C for 5 min, discard the supernatant, and wash the cells twice with PBS. Add 1 mL of PBS to resuspend the cells, divide the cells into two equal groups, and transfer them into 1.5 mL EP tubes. Label the 1.5 mL EP tubes with the group number.

[0032] 3.2.5 Flow cytometry staining (cell membrane staining): The method is the same as 3.2.2.

[0033] 3.2.6 Flow cytometry staining (nuclear staining): The method is the same as 3.2.3.

[0034] 3.3 In vitro study of Dendrobium arytenoides regulating tumor-associated macrophage polarization (1) Extraction of mouse bone marrow macrophages (BMDM): C57BL / 6 mice were killed by cervical dislocation and then soaked in 70% anhydrous ethanol for 1-2 min. Three small dishes were poured with an appropriate amount (5 mL) of PBS. Three napkins were placed in the biosafety cabinet. Scissors, tweezers, and a razor blade were prepared. The mouse was taken out and placed on the napkin. The skin of the mouse's hind legs was cut with scissors, and the skin was separated from the muscle with tweezers. The muscle was removed with a razor blade along the tibia and femur of the hind legs (scrape vertically, not cut) until the hip joint (the muscles at the femur can be cut with scissors, but the blood vessels should be avoided). (Operate on both legs together), cut the connection at the hip joint, remove the femur and tibia, place them in pre-cooled sterile PBS and wash once, continue to remove the muscle with a blade, and cut the connection between the femur and tibia, and the connection between the tibia and ankle, place them in pre-cooled sterile PBS and wash once, place the femur and tibia in new pre-cooled sterile PBS, take a section of leg bone, appropriately cut the epiphyseal ends at both ends, take a 26 G needle syringe to draw 0.5-1 mL PBS from one end (as close to the top as possible) and insert it into the bone marrow cavity to flush the bone marrow. After the leg bone turns white, rinse with the other end (the leg bone can be cut off after it turns white to facilitate flushing the remaining part), use a 1 mL pipette tip to gently pipette and disperse the bone marrow cells until there is no red floc, pass the evenly suspended cells through a 40 μM nylon mesh to remove cell clumps, transfer to a centrifuge tube, 1000 r / min, 4 ℃, centrifuge for 5 minutes, discard the supernatant, and obtain mouse bone marrow-derived macrophages BMDM.

[0035] (2) Polarization of mouse bone marrow-derived macrophages (BMDM): Transfer the cells in the centrifuge tube to a 6-well plate (it is recommended that the 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 Abotek Biotechnology Co., Ltd.) with a final concentration of 20 ng / mL, mix well, and place in an incubator for three days. After three days, wash the cells two to three times with preheated PBS (until the red blood cells are rinsed clean), and replace the medium with DMEM medium (DMEM+10% FBS+1% double antibody+20 ng / mL M-CSF); place in an incubator and continue to culture for three days to obtain BMDM macrophages.

[0036] (3) The regulatory effect of Dendrobium arytenoids on macrophage polarization in vitro: Dendrobium arytenoids were used to intervene in macrophages and cultured in an incubator for 48 h. The expression of M0 macrophage markers F4 / 80, M1 macrophage markers CD86 and M2 macrophage markers CD163 were analyzed by flow cytometry to explore the regulatory effect of Dendrobium arytenoids on macrophage polarization in vitro.

[0037] (4) Tumor-macrophage interaction of arytenoids in a cell co-culture model: mATC tumor cells were inoculated in the upper chamber of Transwell, and M-CSF-induced BMDM macrophages were inoculated in the lower chamber of Transwell to construct a tumor-macrophage co-culture model. The arytenoids solution containing arytenoids was co-incubated with mATC tumor cells in the upper chamber for 48 h. The upper chamber was removed, and the levels of F4 / 80, CD86, and CD163 in BMDM macrophages in the lower chamber were measured by flow cytometry.

[0038] 4. In vitro and in vivo studies on the effects of Dendrobium arytenoides on blocking the immunosuppressive microenvironment and on the function of cytotoxic T cells 4.1 Blocking effect of dendrobium arytenoids on the immunosuppressive microenvironment: Macrophages were treated with dendrobium arytenoids and cultured in an incubator for 48 h. The culture supernatant of macrophages treated with dendrobium arytenoids 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 instructions of the ELISA kit (Wuhan Aibote Biotechnology Co., Ltd.). The effect of dendrobium arytenoids on macrophage cytokine secretion was investigated and the blocking effect of dendrobium arytenoids on the immunosuppressive microenvironment was analyzed.

[0039] 4.2 Effects of Dendrobium arytenoides on cytotoxic T cell function in vitro The plate was coated with CD3 antibody and placed in a refrigerator at 4°C overnight to extract mouse spleen T cells. The cells were stained with CFSE (BioLegend) in the dark (1 uL (1000x) of CFSE can be used for 1 × 10 7) After 20 min, wash twice with PBS and set aside. Use unstained T cells to perform flow cytometry as negative reference in advance, adjust the parameters to the appropriate size (use FITC channel), and then use flow cytometry to detect the stained T cells as positive reference. The remaining stained T cells were inoculated in a 96-well plate. According to different design groups, different concentrations of dendrobium arytenoids were added and co-cultured with T cells. After incubation at 37 ° C for 72 h, the cells were collected, centrifuged at 1000 r / min, 4 ° C for 5 min, and the cells were blocked with 5% BSA solution. After 15 min, centrifuged at 1000 r / min, 4 ° C for 5 min, and the cells were collected and stained with CD3 flow cytometry antibody (BioLegend) (FITC cannot be used, and it needs to be away from the light of CFSE. PE / APC can be used) for 30 min later, the cells were washed twice with PBS and tested on a flow cytometer. The CFSE status was observed in the CD3+ gate to investigate the effect of Dendrobium arytenoides on T cell proliferation. The same method as above was used to coat the plate with CD3 antibodies, extract T cells, and perform CFSE staining. The macrophages treated with Dendrobium arytenoides were co-cultured with T cells. After incubation at 37°C for 72 h, the cells were collected, stained, and flow cytometry analysis was performed in the same manner as above to investigate the changes in cytotoxic T cell function to clarify whether macrophages can affect cytotoxic T cell function through Dendrobium arytenoides.

[0040] Effect of Dendrobium arytenoidin on the immune checkpoint PD-1 immunotherapy in mATC anaplastic thyroid cancer mice Twenty-four 6-week-old C57BL / 6 female mice weighing 17-20 g were randomly divided into a blank control group, a 100 mg / kg group, a 2 mg / kg group, and a 100 mg / kg+2 mg / kg group after adaptive feeding for 7 days, with 6 mice in each group. mATC tumor cells were cultured at a density of 1 × 10 6 A cell suspension of 100 cells / mL was inoculated subcutaneously in the axilla of the right forelimb of mice. Body weight was continuously monitored, and mice were sacrificed after 4 weeks to compare tumor diameters between groups. Tumor tissues were obtained from experimental mice and fixed, dehydrated, embedded, sectioned, and prepared into paraffin sections of mouse lung tissue. HE staining was performed on a blank control group, a arytenoidin group, a PD-1 group, and a arytenoidin + PD-1 combination group to analyze the effect of arytenoidin on the immune checkpoint PD-1 immunotherapy in mice bearing mATC anaplastic thyroid cancer.

[0041] 4.4 Regulation of arytenoids by dendrobium officinale on PD1 and CD152 cells in tumor tissues and spleen tissues of mATC undifferentiated thyroid cancer mice: Flow cytometry was used to detect the number of PD1 and CD152 cells in the tumor tissues and spleen tissues of each group of mice to explore the regulatory effect of dendrobium officinale on PD1 and CD152 cells.

[0042] 5. Study on the effect of Dendrobium arytenoides on the expression of M1 macrophage proteins by regulating SYK 5.1 Network Pharmacology Study of Dendrobium labialisin Against ATC: Potential targets of dendrobium labialisin were identified using the PharmMapper, TCMSP, SEASearchServer, and STITCH online databases. Host targets of ATC were also identified using the GeneCards database. Intersection targets between dendrobium labialisin and ATC were identified using the online program Draw VennyDiagram. A protein-protein interaction (PPI) network was constructed using the STRING database and Cytoscape 3.9.1 software to identify key targets. GO function and KEGG pathway enrichment analysis were performed on these targets.

[0043] 5.2 Molecular docking analysis of the optimal docking target of arytenoidin against ATC: Molecular docking of the core target and small molecules was performed using AutoDockVinav1.2.0 software, and the binding energy and binding mode of arytenoidin with the target protein were analyzed. The docking results were visualized using PyMOLv2.5, and the optimal docking target of arytenoidin against ATC was finally determined.

[0044] 5.3 Effect of Dendrobium arytenoids on SYK protein expression in macrophages: PCR, immunofluorescence and Western blot were used to detect the effect of Dendrobium arytenoids on SYK protein expression in macrophages.

[0045] Study on the mechanism of action of Dendrobium arytenoides in regulating SYK expression and promoting M1 macrophage polarization against anaplastic thyroid cancer 6.1 Dendrobium arytenoids promote M1 macrophage polarization in a SYK-dependent manner: SYK was knocked out in macrophages by transfecting shRNA into macrophages to test the knockdown efficiency of shSYK. After SYK knockdown, dendrobium arytenoids were used to treat macrophages and investigate changes in macrophage function to confirm that the promotion of M1 macrophage polarization by dendrobium arytenoids is SYK-dependent.

[0046] 6.2 Dose-effect relationship of arytenoids in inducing or inhibiting SYK expression in macrophages: By administering different concentrations of arytenoids at different time points to macrophages, Western blot was used to investigate the changes in SYK expression in macrophages, confirming that there was a dose-effect relationship between arytenoids inducing or inhibiting SYK expression. 6.3 Dendrobium arytenoids induce or inhibit SYK expression in macrophages 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 establish a tumor-macrophage co-culture model. Tumor cells were treated with different concentrations of Dendrobium arytenoids at different time points and incubated for 48 hours. Changes in macrophage SYK expression were examined by Western blot.

[0047] Example 2: Result Analysis 1. Dendrobium officinale extract inhibits ATC cell activity CCK8 assay detected the effects of a series of compounds on the proliferation of ATC cell line 8505C. The CCK8 results showed that arytenoidin had a strong inhibitory effect on ATC cells ( Figure 1 ).

[0048] 2. Effects of Dendrobium arytenoides on tumor cell proliferation and cloning To investigate the effect of arytenoids on tumor cell proliferation, CCK8 was used to detect the effect of arytenoids on the proliferation of ATC cell line 8505C cells after 48 h. The plate cloning ability of arytenoids on ATC cell line 8505C was evaluated by plate cloning. The CCK8 results showed that arytenoids could inhibit the proliferation of ATC cell line 8505C ( Figure 2 A) Cell proliferation. Further plate cloning experiments showed that Dendrobium arytenoids significantly inhibited the proliferation of ATC cell line 8505C ( Figure 2 B) Plate cloning ability of cells.

[0049] 3. Effect of Dendrobium arytenoides on cell apoptosis The results of flow cytometry apoptosis assay showed that dendrobium arytenoids could significantly induce apoptosis of ATC cells ( Figure 3 ).

[0050] 4. Dendrobium officinale inhibits ATC cell cycle The effect of arytenoids on the ATC cell cycle was detected by flow cytometry. The results showed that arytenoids could cause cell cycle arrest in ATC cells 8505C and reduce the ratio of G0 / G1 phase cells ( Figure 4 ).

[0051] 5. Inhibitory effect of Dendrobium arytenoides on ATC tumor growth The tumor transplantation experiment in C57BL / 6 female mice showed that Dendrobium arytenoids significantly inhibited the growth of ATC cell tumors, while there was no significant change in the body weight of the mice ( Figure 5AB). After reaching the end point of the experiment, the mice were killed and the heterotopic transplanted tumors were removed. It was found that the tumor volume and tumor weight of the arytenoid group (100 mg / Kg) were significantly reduced ( Figure 5 CD).

[0052] 6. Regulation of Dendrobium arytenoids on macrophages, MDSCs, T cells, and NK cells in tumor tissues and spleen tissues of MATC undifferentiated thyroid cancer mice Flow cytometry analysis was performed to investigate the regulatory effects of arytenoid dendrobium on macrophages, T cells, NK cells, and MDSC cells in tumor tissues and spleen tissues of MATC undifferentiated thyroid cancer mice. The flow cytometry results of tumor tissues showed that arytenoid dendrobium upregulated M1 macrophages. Compared with the control group, the arytenoid dendrobium group had a statistically significant effect on the increase in M1 macrophages ( Figure 6 -A1, P <0.05); Dendrobium arytenoids administration down-regulated M2 macrophages, and the effect of Dendrobium arytenoids group on the reduction of M2 macrophages was statistically significant compared with the control group ( Figure 6 -A1, P <0.05); Dendrobium officinale administration increased CD8 cells, and the effect of Dendrobium officinale administration on CD8 cells was statistically significant compared with the control group ( Figure 6 -A6, P <0.001); The flow cytometry results of spleen tissue showed that the administration of arytenoid dendrobium upregulated M1 macrophages. Compared with the control group, the arytenoid dendrobium group had a statistically significant effect on the increase of M1 macrophages ( Figure 6 -B1, P <0.05); Dendrobium arytenoids administration down-regulated M2 macrophages, and the effect of Dendrobium arytenoids group on the reduction of M2 macrophages was statistically significant compared with the control group ( Figure 6 -B1, P <0.05); Dendrobium lipoides administration upregulated MDSC cells, and the effect of Dendrobium lipoides administration on the increase of MDSC cells was statistically significant compared with the control group ( Figure 6 -B2, P <0.01); Dendrobium arytenoids administration upregulated M-MDSC cells, and the effect of Dendrobium arytenoids group on the increase of M-MDSC cells was statistically significant compared with the control group ( Figure 6 -B3, P <0.001); Dendrobium arytenoids administration upregulated PMN-MDSC cells, and the effect of Dendrobium arytenoids group on the increase of PMN-MDSC cells was statistically significant compared with the control group ( Figure 6 -B4, P <0.05).

[0053] 7. Regulatory effects of Dendrobium arytenoides on macrophage polarization In order to explore the regulatory effect of Dendrobium arytenoids on macrophage polarization in vitro, Dendrobium arytenoids were used to intervene in mouse bone marrow-derived macrophages BMDM. The expression of M0 macrophage markers F4 / 80, M1 macrophage markers CD86 and M2 macrophage markers CD163 were analyzed by flow cytometry. The flow cytometry results showed that Dendrobium arytenoids promoted the high expression of M1 macrophage marker CD86 and inhibited the expression of M2 macrophage marker CD163 ( Figure 7 AB).

[0054] 8. Effect of Dendrobium officinale extract on T cell proliferation In order to explore the effect of Dendrobium officinale on T cell proliferation in vitro, a Dendrobium officinale-T cell co-culture model was used to investigate the effect of Dendrobium officinale on T cell proliferation. Figure 8 AB). Flow cytometry results showed that Dendrobium arytenoides could not promote T cell proliferation.

[0055] 9. Exploring the key targets of Dendrobium arytenoides in regulating the anti-ATC effect of tumor-associated macrophages based on network pharmacology, molecular docking, and kinetic experiments Using network pharmacology, it was preliminarily determined that arytenoid dendrobium can achieve anti-ATC effects by acting on the host, multiple targets, and multiple pathways. The results showed that arytenoid dendrobium has 57 potential anti-ATC targets ( Figure 9 AB), HSP90AA1, ESR1, ERBB2 and MMP9 are the potential core targets of Dendrobium arytenoids to exert anti-ATC effects ( Figure 9 C), using the TISCH database, we created a visualization of SYK expression in various cell types of thyroid cancer, potentially indicating that SYK is an important target of Dendrobium arytenoids in regulating macrophage M1 polarization against ATC ( Figure 9 D), molecular docking binding kinetics experiments were used to analyze the binding effect of arytenoids and SYK. The results showed that KSYK formed two hydrogen bonds with the amino acid residue THR-64 in arytenoids and formed a pi-cation conjugation with the amino acid residue TYR-63 in arytenoids ( Figure 9 E).

[0056] 10. Based on immunofluorescence experiments, it was verified that SYK is the key target of Dendrobium arytenoids in regulating the anti-ATC of tumor-associated macrophages Immunofluorescence experiments were used to verify whether SYK was co-expressed on ATC. The results showed that SYK was co-expressed on ATC ( Figure 10 ).

[0057] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. Use of a dendrobium arytenoidin in the preparation of immunotherapeutic drugs against anaplastic thyroid cancer.

2. The use according to claim 1, characterized in that Dendrobium arytenoides inhibits the growth of anaplastic thyroid cancer cells in vivo.

3. The use according to claim 1, characterized in that Dendrobium arytenoids inhibit the progression of anaplastic thyroid cancer by promoting M1 macrophage polarization through regulating spleen tyrosine kinase.

4. An immunotherapy drug for anaplastic thyroid cancer, characterized in that: A therapeutically effective amount of arytenoidin is included.

5. Use of spleen tyrosine kinase as a target for arytenoid dendrobium in the treatment of anaplastic thyroid cancer.

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