Application of Rhodiolin in the preparation of drugs for treating human papillary thyroid carcinoma

Abstract

The present invention relates to a new use of Rhodiolin, and discloses the use of Rhodiolin in preparing a drug for treating human thyroid papillary carcinoma. The present invention proves through in vivo and in vitro experiments that Rhodiolin can effectively treat human thyroid papillary carcinoma, inhibit tumor cell proliferation and induce apoptosis, and downregulate the expression of cyclin in thyroid papillary carcinoma cells. At the same time, it promotes apoptosis, indicating that Rhodiolin has a good effect of resisting human thyroid papillary carcinoma. In addition, Rhodiolin has the advantages of no toxic side effects, small dosage, wide source of raw materials, low production cost, etc.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of Rhodiolin in preparing medicine for treating human papillary thyroid carcinoma. Technical Background

[0002] Papillary thyroid carcinoma originates from thyroid epithelial cells, and its pathological features are typical papillary or follicular. It is a common endocrine malignancy. Papillary thyroid carcinoma accounts for more than 80% of all thyroid cancer incidences. The 5-year survival rate of advanced thyroid cancer worldwide is only 59%. In the past 20 years, the incidence of papillary thyroid carcinoma has increased 20 times. A study based on the latest global cancer burden data in 2020 showed that papillary thyroid carcinoma ranks 9th in incidence among men worldwide and 4th in incidence among women worldwide. In some countries, such as South Korea and Japan, the incidence of papillary thyroid carcinoma has jumped to the second place among all cancers in women. Papillary thyroid carcinoma has had a great impact on human health in the world and has placed a heavy burden on the public health system.

[0003] It is worth noting that although papillary thyroid cancer can usually be successfully treated with surgical resection and the patient has a good prognosis, the cancer cells of advanced papillary thyroid cancer will rapidly proliferate and metastasize to multiple organs such as bones, lungs, and cervical lymph nodes in a short period of time. Most papillary thyroid cancers occur in the thyroid lobe, and a few accumulate in the isthmus of the thyroid. It is reported that the incidence of papillary thyroid cancer in the isthmus of the thyroid is about 19.2%. Compared with cancers located in other parts of the thyroid gland, papillary thyroid cancer originating from the isthmus will invade the thyroid capsule and adjacent tissues, and lead to an increased rate of central lymph node involvement.

[0004] The occurrence of papillary thyroid carcinoma involves multiple factors and multiple links, including oncogene mutations (BRAF mutations, RAS mutations, and PET / PTC and TRK rearrangements, etc.), abnormally elevated thyroid stimulating hormone (thyrotropin, TSH) levels, ionizing radiation, iodine deficiency or high iodine, genetic factors, etc. Although the incidence of papillary thyroid carcinoma has increased year by year, the diagnosis, classification and treatment options for early and late papillary thyroid carcinoma are still limited. At present, there is no recognized effective treatment for papillary thyroid carcinoma in clinical practice, and the clinical treatment of papillary thyroid carcinoma is still mainly surgical resection plus radioactive iodine 131 treatment. It cannot be ignored that surgical resection of the thyroid gland will destroy the homeostasis of thyroid hormone levels in the body, seriously affect the levels of other hormones in the body, and patients need to take thyroid hormones for life. In recent years, targeted drugs have gradually been used in the treatment of papillary thyroid carcinoma. In 2017, the multikinase inhibitor Sorafenib was approved by CFDA as a treatment for papillary thyroid carcinoma. It is also the only targeted drug for radioactive iodine-refractory differentiated papillary thyroid carcinoma so far. However, this drug molecule is an inhibitor of tyrosine kinase, which can simultaneously inhibit multiple targets such as VEGF1-3, PDGF, c-Kit, etc., thereby achieving the effect of inhibiting tumor growth. Due to the lack of target selectivity, the drug efficacy is limited, and it has adverse reactions such as skin and mucosal reactions, diarrhea, hypertension and fatigue, resulting in poor patient tolerance, high rates of reduction and discontinuation, and inability to continue medication, limiting the benefits. With the elucidation of the pathogenesis of papillary thyroid carcinoma, gene inhibitor drugs have gradually been used in the clinical treatment of papillary thyroid carcinoma. For example, the BRAF gene inhibitor dabrafenib is a new type of targeted drug that has significant anti-tumor activity in the treatment of BRAF-positive ATC patients, but tumor cells are prone to develop resistance to it within 6-7 months of treatment. At present, both traditional chemotherapy drugs and new targeted drugs have certain side effects and tumor resistance. Therefore, in-depth revelation of potential mechanisms and targeted development of targeted new drugs are crucial to improve patients' overall survival rate and improve their quality of life.

[0005] Rhodiolin is a natural flavonoid compound extracted from the medicinal plant Rhodiola rosea. It has a molecular weight of 480.42 and is a yellow powder. It is water-soluble and has no toxic side effects. There are reports that it has the effect of protecting myocardial cells in vivo, but there are no reports on its anti-tumor effects. Summary of the invention

[0006] Purpose of the Invention

[0007] The object of the present invention is to provide a new use of Rhodiolin.

[0008] Technical Solution

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] The use of Rhodiolin in the preparation of a drug for treating human papillary thyroid carcinoma, wherein the structural formula of Rhodiolin is as follows:

[0011]

[0012] In the technical solution of the present invention, the dosage forms of Rhodiolin include oral preparations and injection preparations.

[0013] Beneficial Effects

[0014] Papillary thyroid carcinoma has the characteristics of high morbidity, low mortality and low metastasis rate. Although traditional surgical resection, radiotherapy and other methods have made significant progress in the treatment of papillary thyroid carcinoma, these methods are too harmful to patients. Therefore, we urgently need a drug that is different from the one used to treat highly lethal tumors, hoping to inhibit the proliferation of papillary thyroid cancer cells and induce their apoptosis in a non-invasive and side-effect-free way, so that the tumor tissue is gradually ablated, and the purpose of non-invasive treatment is achieved.

[0015] Most chemical anti-tumor drugs have side effects, such as inhibiting normal cells. The present invention is the first to discover that Rhodiolin has a special mechanism, that is, it has a good therapeutic effect on human papillary thyroid carcinoma and has no inhibitory effect on normal cells.

[0016] Specifically: For example, the normal thyroid follicular epithelial cell Nthy-ori 3-1 has no significant change in cell viability within 80 μM, and its ultra-high dose is safe for Nthy-ori 3-1 cells, and the highest dose in subsequent experiments is 40 μM. Rhodiolin has a significant inhibitory effect on TPC-1 cell proliferation; Rhodiolin blocks the cell cycle of TPC-1 and BCPAP cells, that is, Rhodiolin treatment reduces the expression of cell cycle proteins in papillary thyroid cancer cells in a dose-dependent manner, and increases the expression of cell cycle protein kinase inhibitors. Rhodiolin treatment at the experimental concentration significantly promotes apoptosis of papillary thyroid cancer cells, but does not induce apoptosis of normal thyroid epithelial cells. Rhodiolin treatment increases the production of active spliceosomes of Caspase-3, the most important terminal cleavage enzyme for apoptosis, in a dose-dependent manner. The present invention proves through in vitro and in vivo experiments that Rhodiolin can effectively treat human papillary thyroid carcinoma, inhibit tumor cell proliferation and induce apoptosis, and downregulate the expression of cyclins in papillary thyroid cancer cells. At the same time, it promotes apoptosis, indicating that Rhodiolin has a good anti-human papillary thyroid carcinoma effect. Therefore, we found that Rhodiolin has a good therapeutic effect on papillary thyroid carcinoma both in vivo and in vitro, which will provide a new option for the treatment of papillary thyroid carcinoma, which is beneficial to improve the quality of life of patients and reduce the pressure on the public health system caused by the increasing incidence of papillary thyroid carcinoma.

[0017] In addition, Rhodiolin has the advantages of no toxic side effects, small dosage, wide source of raw materials, and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To evaluate the safety of Rhodiolin on normal thyroid epithelial cells Nthy-ori 3-1 cells.

[0019] Figure 2 CCK-8 assay for Rhodiolin's inhibition of thyroid papillary carcinoma cell activity, A. TPC-1 cells; B. BCPAP cells.

[0020] Figure 3 The results are for bright field.

[0021] Figure 4 These are the results of EdU staining when the Rhodiolin concentrations are 0μM, 10μM, 20μM, and 40μM. A. EdU staining fluorescence images of TPC-1 and BCPAP cells after Rhodiolin treatment; B. Statistics of the red fluorescence ratio in Figure A.

[0022] Figure 5Flow cytometric detection of the cell cycle of TPC-1 and BCPAP cells after Rhodiolin treatment. A. Cell cycle distribution histogram; B. Statistics of the proportion of each cell cycle shown in Figure A.

[0023] Figure 6 The effect of Rhodiolin on the expression of cell cycle-related genes in thyroid papillary carcinoma cells. A. Western Blot analysis of the protein expression levels of cell cycle-related genes CDK2, CDK4, p21, p27, Cyclin A, and Cyclin E in thyroid papillary carcinoma cells treated with different concentrations of Rhodiolin; B. Statistics of Figure A.

[0024] Figure 7 Figure 1 is a graph showing the effect of different concentrations of Rhodiolin on apoptosis of thyroid papillary carcinoma TPC-1 and BCPAP cells and normal thyroid epithelial cells Nthy-ori 3-1 cells detected by flow cytometry; B. is a statistical analysis of the results in Figure A.

[0025] Figure 8 The effect of Rhodiolin on the expression of Caspase-3, the most important terminal cleavage enzyme in thyroid papillary cell apoptosis. A. Western Blot analysis of the effect of different concentrations of Rhodiolin on the expression of Caspase-3, the most important terminal cleavage enzyme in thyroid papillary cell apoptosis; B. Statistics of Figure A.

[0026] Fig. 9 Effects of Rhodiolin treatment on tumor growth in vivo, A. In vivo imaging; B. Food and water intake; C. ROI fluorescence intensity statistics; D. Body weight.

[0027] Fig.10 HE, Ki-67 and TUNEL staining of tumor tissues.

[0028] Fig.11 Serum AST and ALT were detected in mice after Rhodiolin treatment.

[0029] Fig.12 HE staining of major tissues was performed to evaluate the safety of the drug in vivo. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto:

[0031] Example 1 Rhodiolin inhibits proliferation of human papillary thyroid carcinoma cells in vitro

[0032] 1. Drug treatment of TPC-1 cells, BCPAP cells and Nthy-ori 3-1 cells

[0033] TPC-1 cells, BCPAP cells and Nthy-ori 3-1 cells were purchased from the cell bank of Xiangya School of Medicine, Central South University. Rhodiolin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. as a white powder with a purity of HPLC greater than or equal to 98%. TPC-1 cells and Nthy-ori 3-1 cells were cultured at 37°C, 5% CO2 by volume, and 10% FBSDMEM by volume. Different concentrations of Rhodiolin were added as drug treatment groups, and TPC-1 cells without drugs were set as control groups. Cell samples were collected 12 hours after drug treatment.

[0034] 2. CCK-8 to evaluate the safety of Rhodiolin

[0035] CCK-8 kit was purchased from Nanjing Jiancheng Bioengineering Institute. In order to evaluate the safety of Rhodiolin, normal thyroid epithelial cells Nthy-ori 3-1 cells were selected as a model in this study. Nthy-ori 3-1 cells were cultured at 1×10 4 The cells were inoculated into a 96-well plate at a density of 100 / well, pre-cultured in an incubator for 24 hours, and serum-free culture medium containing different concentrations of Rhodiolin (0, 5, 10, 20, 40, 80 μM) was prepared, and then the prepared culture medium was added to the 96-well plate and incubated for 12 hours. After the incubation, 10 μl of CCK-8 solution was added to each well of the plate, and the culture plate was placed in the incubator for another 1 hour. The absorbance at 450 nm was measured using an enzyme reader to detect cell viability.

[0036] 3.CCK-8 detection of the half inhibitory concentration of Rhodiolin on the proliferation inhibition of TPC-1 cells and BCPAP cells. In this study, TPC-1 cells were selected as a model, and TPC-1 cells and BCPAP cells were inoculated into 96-well plates at a density of 5000 cells / well. The cells were pre-cultured in an incubator for 24 hours, and serum-free culture medium containing different concentrations of Rhodiolin (0, 5, 10, 20, 40, 80 μM) was prepared. Then, the above-prepared culture medium was added to the 96-well plate and incubated for 12 hours. After the incubation, 10 μl of CCK-8 solution was added to each well of the plate, and the culture plate was placed in the incubator for another 1 hour. The absorbance at 450 nm was measured using an enzyme reader. The cell survival rate was calculated according to the following formula:

[0037] Cell survival rate (%) = [(As-Ab) / (Ac-Ab)]*100

[0038] Inhibition rate (%) = [(Ac-As) / (Ac-Ab)]*100

[0039] As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 and test compounds);

[0040] Ab = absorbance of blank well (absorbance of wells containing culture medium and CCK-8);

[0041] Ac = absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8).

[0042] 4. Flow cytometry to detect the effect of Rhodiolin on apoptosis in human papillary thyroid carcinoma cells

[0043] Carefully collect the cell culture medium into a centrifuge tube for later use. Digest the cells with EDTA-free trypsin until the cells can be gently blown down with a pipette or gun tip, then add the previously collected cell culture medium, blow down all the adherent cells, and gently blow away the cells. Collect again into a centrifuge tube. Centrifuge at 1000rpm for 6 minutes to precipitate the cells. Carefully aspirate the supernatant, leaving about 50μl of culture medium to avoid aspirating the cells. Add about 1ml of 4℃ pre-cooled PBS, resuspend the cells, centrifuge again to precipitate the cells, carefully aspirate the supernatant, dilute the binding buffer with deionized water at a ratio of 1:3 (4ml 4x binding buffer + 12ml deionized water); resuspend the cells with 1x binding buffer and adjust its concentration to 1-5*10 6 / ml; take 100μl of cell suspension in a 5ml flow tube, add 5μl Annexin V / Alexa Fluor 647 and 10μl 20μg / ml propidium iodide solution; mix well and incubate at room temperature in the dark for 10 minutes and then immediately perform flow cytometry detection.

[0044] 5. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)

[0045] TRIzol was used to lyse cells and extract total RNA. Reverse transcription was performed using the Takara reverse transcription kit. Primers for the target gene were designed according to the Primer3 software, and then synthesized by Nanjing Qingke Biotechnology Co., Ltd. The reaction system was prepared according to the instructions of the Vazyme kit of Nanjing Novozyme Biotechnology Co., Ltd., and then the amplification reaction was performed on a qPCR instrument. The relative expression of the target gene was calculated based on the Ct value.

[0046] 6. Western Blot Analysis

[0047] After drug treatment of TPC-1 cells and BCPAP cells, cells were cultured in 6 cm dishes, 300 μL of lysis buffer was added to each well, and after sufficient cell lysis, the cells were centrifuged at 4 ° C and 14000 rpm for 10 min, and the supernatant was collected. The protein concentration was determined using the Biyuntian BCA assay kit. After adjusting the protein concentration, it was mixed with 6×Sample buffer and placed in a 100 ° C dry bath for 7 min to completely denature the protein. Finally, the extracted protein sample was stored in a -80 ° C refrigerator for later use. Using SDS-PAGE gel electrophoresis with a mass percentage of 10% or 12%, the target band was transferred to a PVDF membrane and blocked at room temperature for 1 h in a mass percentage of 5% skim milk. Incubate the primary antibody at 4 ° C overnight. After incubation, wash 3 times with TBST for 10 min each time. After incubating the secondary antibody for 1 h at room temperature, wash 3 times with TBST for 10 min each time. The PVDF membrane was immersed in the colorimetric solution, and the target protein band was observed by the Tanon5200 chemiluminescence imaging system.

[0048] Effects on ROS levels in TPC-1 cells and on the proliferation of TPC-1 cells.

[0049] 7. EdU Staining

[0050] TPC-1 cells and BCPAP cells were seeded into 6-well plates. After the cells were cultured for 12 hours and adhered to the wall, 0, 10, 20 and 40 μM Rhodiolin were added for 12 hours. The culture medium was discarded and the cells were gently shaken and washed once with PBS to prevent the cells from falling off. The cells were stained using the RIBOBIO Cell-LightTM EdU Apollo In Vitro Kit. EdU solution (reagent A) was diluted with complete cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU culture medium. 100 μl of 50 μM EdU culture medium was added to each well and incubated for 2 hours. The culture medium was discarded and the cells were washed with PBS 1 to 2 times, each for 5 minutes. Then, the cells were fixed with PBS containing 4% paraformaldehyde at room temperature for 30 minutes. After fixation, the cells were washed with 2 mg / ml glycine solution, PBS and PBS containing 0.5% TritonX-100 for 5 minutes each. Finally, Apollo staining and DNA staining (Hoechest 33342 staining or DAPI staining) were performed and the fluorescence was observed and photographed under LSCM.

[0051] 8. Flow cytometry to detect cell cycle

[0052] TPC-1 cells were inoculated into 6-well plates. After the cells were cultured for 12 hours and attached to the wall, 0, 150 and 300 μM Rhodiolin were added for 24 hours. The culture medium was discarded and washed once with PBS to prevent the cells from falling off. Trypsin was added for digestion. After digestion was complete, complete culture medium was added to terminate digestion and washed once with pre-cooled PBS. Then 1 ml of 70% ethanol pre-cooled in an ice bath was added to fix overnight at -20°C, centrifuged at 600g / min for 10 minutes to remove the ethanol, washed with PBS, and centrifuged at 600g / min for 10 minutes to remove the PBS. 100 μl of RNaseA was added to fully suspend the cells and bathed at 37°C for 30 minutes. Then, 400 μl of propidium iodide (PI) solution was added and mixed thoroughly, and incubated at 2-8°C in the dark for 30 minutes. Finally, red fluorescence (PE channel) was detected by flow cytometry at an excitation wavelength of 488nm.

[0053] 9. Experimental Results

[0054] 9.1 Safety evaluation of rhodiolin on normal thyroid follicular epithelial cells

[0055] like Figure 1 As shown, Rhodiolin treatment of Nthy-ori 3-1 cells for 12 hours had no significant change in cell viability within 80 μM, so the dose of Rhodiolin used in this study is safe for Nthy-ori 3-1 cells.

[0056] 9.2 Rhodiolin inhibits CCK-8 detection of papillary thyroid carcinoma

[0057] like Figure 2 As shown in the figure, with the increase of Rhodiolin administration concentration, the viability of TPC-1 and BCPAP cells decreased in a dose-dependent manner. Figure 3 It is the result of bright field.

[0058] 9.3 EdU / DAPI double staining shows the inhibitory effect of Rhodiolin on TPC-1 and BCPAP cell proliferation Figure 4 As shown in the figure, when the concentration of Rhodiolin was 0μM, 10μM, 20μM, and 40μM, the percentage of EdU staining positive cells decreased in a dose-dependent manner. Therefore, Rhodiolin has a significant inhibitory effect on the proliferation of TPC-1 cells.

[0059] 9.4 Rhodiolin treatment arrests the cell cycle of TPC-1 and BCPAP cells

[0060] like Figure 5As shown, when the concentration of Rhodiolin was 0μM, 10μM, 20μM, and 40μM, the percentage of S phase cells of TPC-1 and BCPAP cells decreased in a dose-dependent manner, and the number of G0G1 phase cells increased in a dose-dependent manner. The DMSO used to dissolve the drug in the experiment had no effect on the cell cycle results after dilution. This shows that Rhodiolin blocked the cell cycle of TPC-1 and BCPAP cells.

[0061] 9.5 Western Blot analysis of the effect of Rhodiolin treatment on the expression of cell cycle-related proteins in papillary thyroid carcinoma cells Figure 6 As shown, rhodiolin treatment dose-dependently decreased the expression of cell cycle proteins and increased the expression of cyclin kinase inhibitors in papillary thyroid carcinoma cells.

[0062] 9.6 Rhodiolin treatment promotes apoptosis of papillary thyroid carcinoma cells

[0063] like Figure 7 As shown, flow cytometry was used to detect the effects of different concentrations of Rhodiolin on apoptosis of thyroid papillary carcinoma TPC-1 and BCPAP cells and normal thyroid epithelial cells Nthy-ori 3-1. The results showed that Rhodiolin treatment at the experimental concentration significantly promoted apoptosis of thyroid papillary carcinoma cells, but did not induce apoptosis of normal thyroid epithelial cells.

[0064] 9.7 Western Blot analysis of the effect of Rhodiolin treatment on the expression of apoptosis-related proteins in papillary thyroid carcinoma cells Figure 8 As shown, Rhodiolin treatment increased the production of active cleavage forms of Caspase-3, the main terminal cleavage enzyme involved in apoptosis, in a dose-dependent manner.

[0065] Example 2 Application of Rhodiolin in the Treatment of Orthotopic Xenograft Tumor Model Mice of Human Thyroid Papillary Carcinoma 1. The experimental animals selected were 6-week-old male C57BL / c mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and were raised according to the standard feeding process for mice (National Institutes of Health and China Pharmaceutical University Laboratory Animal Feeding and Use Guidelines). All mice were raised in a standard environment of 12 hours of light / 12 hours of darkness, constant temperature and humidity, and after the mice adapted to the environment, the subsequent related experiments were performed. All experiments have been approved and carried out in accordance with the guidance of the Experimental Animal Management Committee of China Pharmaceutical University (License No. SYXK-2021-0011). D-Luciferin was purchased from Sigma.

[0066] 2. TPC-1-Luc2 cell construction materials: strains and plasmids DH5α competent Escherichia coli was purchased from Nanjing Novozyme Biotechnology Co., Ltd., China, pMX vector, pVSVg plasmid and psPAX2 plasmid were purchased from Addgene, USA, and pGL4.17 plasmid was purchased from Clontech. Cells and experimental animals 293T gag-pol cell line and BALB / c nude were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., female, 6 weeks old, weighing 17-19g. Reagents and instruments Taq DNA polymerase, T4 DNA ligase, HindIII, XbaI, EcoRI, NotI were purchased from NEB.

[0067] 2.2 Methods

[0068] 1.2.1 Construction of pMX-Luc2. The pGL4.17[Luc2 / neo] plasmid was digested with HindIII, pCMV was amplified from the PCI-neo vector, and the two were ligated with ligase. The ligation product was transformed, clones were selected for sequencing identification, and the correctly identified pGL4.17[Luc2 / pVSVg] and pMX were double-digested with EcoRI and NotI. The digested Luc2 / pCMV was ligated with the pMX large fragment to form pMX-Luc2, which was sent for sequencing identification.

[0069] 2.2.2 Co-transfection of recombinant plasmids into 293T cells. According to the instructions of Lipofectamine 3000TM, the pMX-Luc2 cloned plasmid identified by sequencing was co-transfected into 293T cells with pVSVg plasmid and psPAX2 plasmid. After 6 hours of transfection, 2 ml of fresh DMEM complete medium at 37°C was added.

[0070] 2.2.3 Harvesting of Luc2-expressing lentivirus. 48 hours after transfection, collect the culture supernatant containing retrovirus and add fresh DMEM complete culture medium to continue culturing. After 48 hours, collect the lentiviral supernatant and cells, freeze-thaw repeatedly in liquid nitrogen for 3 times to lyse the cells and release the virus. Concentrate the lentivirus collected several times and store it at -80℃.

[0071] 2.2.4 Establishment and screening of Luc2-positive TPC-1 cell lines. 100 μL of lentiviral supernatant expressing Luc2 was inoculated into the human papillary thyroid cancer cell line TPC-1 cells. Infect once every 12 hours for a total of 4 times. After culture, add 1 μl of 15 mg / ml D-luciferin to each well, gently shake the cell plate to evenly distribute it in the culture medium, and immediately detect it in the in vivo imaging system. Pick the positive clones and expand the culture.

[0072] 2.2.5 Detection of luciferase expression in TPC-1-Luc2 cells by in vivo imaging system. 7 TPC-1-Luc2 cells with a concentration of 100 μl / ml were injected into BALB / c nude mice through the tail vein, with 2 nude mice injected with 100 μl and the other 2 mice injected with 300 μl. D-Luciferin (150 mg / kg) and sodium pentobarbital (55 mg / kg) were intraperitoneally administered 1 hour and 72 hours after tumor cell inoculation, and the expression of luciferase was observed under a live imaging system 10 minutes after injection.

[0073] 3. HE, Ki-67 and TUNEL staining of tumor tissue

[0074] After the administration, the tumor tissues were collected and stained according to the standard experimental methods of HE, Ki-67 and TUNEL staining.

[0075] 4. HE staining of major tissues for drug safety evaluation

[0076] After the administration, the heart, liver, spleen, lung and kidney tissues were obtained and stained according to the standard experimental method of HE staining.

[0077] 5. Serum AST, ALT detection

[0078] After administration, blood was collected, placed at 4 degrees Celsius overnight, and then centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and serum AST and ALT were tested according to the instructions.

[0079] 6. Experimental Results

[0080] 6.1 In vivo imaging

[0081] like Fig. 9 As shown in Figure 6, Rhodiolin treatment significantly inhibited tumor growth and induced gradual tumor ablation compared with the control group. Fig.10 As shown, HE staining of the Rhodiolin treatment group showed that the tumor cells were loose and not dense. Rhodiolin treatment reduced the number of Ki-67 positive cells in a dose-dependent manner, and TUNEL staining of tumor tissues showed that Rhodiolin increased tumor tissue cell apoptosis in a dose-dependent manner.

[0082] 6.3 Serum AST and ALT testing

[0083] like Fig.11 As shown, the serum AST and ALT levels of mice in the experimental and control groups were within the normal range.

[0084] 6.4 HE staining of major tissues

[0085] like Fig.12 As shown in the figure, (1) the myocardial fibers are evenly colored, the cell boundaries are clear and the shape is consistent. The myocardial cell striations are clear, with bright and dark opacities alternating. There is no abnormality in the interstitium. No obvious inflammatory changes were observed. (2) The hepatocytes are round and plump, the hepatic plates are arranged regularly and neatly, and the hepatic sinusoids are not obviously expanded or squeezed. No obvious inflammatory changes were observed. (3) The splenic tissue capsule is composed of dense connective tissue rich in elastic fibers and smooth muscle fibers of uniform thickness. The capsule connective tissue extends into the spleen to form trabeculae, with no obvious abnormalities. (4) The splenic substance is red pulp and white pulp. The white pulp is composed of lymphatic sheaths, lymphatic nodules and marginal zones around the central artery. The structure of each layer is clear, and there is no obvious change in quantity and size. The red pulp is distributed in a large area under the capsule, around the trabeculae and outside the marginal zone of the white pulp. It is composed of splenic cords and splenic sinusoids, which are evenly distributed and have no clear boundary with the white pulp. No obvious abnormalities were observed.

[0086] (5) The surface of the lung tissue is covered with a smooth serous membrane with no obvious abnormalities. The lung parenchyma is composed of the branches of the bronchus at all levels and a large number of alveoli at their terminals. The structures of the bronchus at all levels have no obvious abnormalities. The alveolar wall is composed of a single layer of epithelium with a clear structure. The interstitium includes the connective tissue and trachea in the lungs, and there are no obvious abnormalities. No obvious inflammatory changes were observed.

[0087] (6) The surface capsule of the renal tissue is composed of dense connective tissue of uniform thickness: the renal parenchyma is composed of the superficial cortex and deep medulla, with a clear boundary between the cortex and medulla; the glomeruli are evenly distributed in the cortex, the number of cells and matrix in the glomeruli are uniform, the renal tubular epithelial cells are round and plump, the brush borders are arranged neatly and regularly, and no obvious abnormalities are found in the medulla; the connective tissue between the urinary tubules is the renal interstitium, which has no obvious hyperplasia and no obvious inflammatory changes. Therefore, it shows that Rhodiolin has good safety.

Claims

1. The use of Rhodiolin in the preparation of a drug for treating human papillary thyroid carcinoma, characterized in that: The structural formula of Rhodiolin is shown below:

2. The use according to claim 1, characterized in that: The administration forms of Rhodiolin include oral preparations and injection preparations.

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