Traditional Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer as well as preparation method and application of traditional Chinese medicine compatibility formula extract

Abstract

The invention discloses a traditional Chinese medicine compatibility formula composition for treating iodine-refractory thyroid cancer as well as a preparation method and application thereof, and relates to the field of medicines. The traditional Chinese medicine compatibility formula composition is prepared from traditional Chinese medicine raw materials through solvent extraction, and the traditional Chinese medicine raw materials comprise radix paeoniae alba, honey-fried licorice roots, airpotato yam rhizomes, astragalus membranaceus and roasted rhizoma atractylodis macrocephalae. The traditional Chinese medicine compatibility formula extract provided by the invention can reverse the iodine intake effect of iodine refractory differentiated thyroid cancer, has the effect of inhibiting malignant biological behaviors on the iodine refractory differentiated thyroid cancer, and promotes ferroptosis by promoting and inhibiting GPX4 expression, so that the antitumor activity is improved. Meanwhile, the traditional Chinese medicine composition has the effects of cooling blood and decreasing internal heat, emaciating and detoxifying, reducing phlegm and resolving masses, is relatively light in damage degree to the liver and small in side effect, and can reduce the hepatotoxicity of the airpotato yam and enhance the anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of drugs, and particularly to a traditional Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer, a preparation method thereof, and an application thereof. Background Art

[0002] Radioiodine-refractory differentiated thyroid cancer (RAIR-DTC) is a subtype of differentiated thyroid cancer (DTC) in which the lesions still do not take up iodine or the disease progresses after radioactive iodine-131 treatment, accounting for 5%-23% of patients with differentiated thyroid cancer. Differentiated thyroid cancer includes papillary carcinoma and follicular carcinoma, accounting for more than 90% of thyroid cancer. Distant metastasis is the main cause of death in patients with differentiated thyroid cancer. Among them, about 1 / 3 of the patients with distant metastasis will develop into radioiodine-refractory differentiated thyroid cancer, and its 10-year survival rate is only 10%.

[0003] Among them, the sodium-iodide symporter (NIS) is a marker protein of thyroid cells, and its core function is to actively transport iodide ions (I-) from outside the cell to inside the cell to provide raw materials for the synthesis of thyroid hormones (T3, T4). The expression level of NIS directly determines the iodine uptake ability of thyroid cells and is the key molecular basis for radioactive iodine therapy (RAI). In iodine-refractory thyroid cancer (IRTC), the expression of NIS protein is significantly reduced or absent, and its mechanism involves abnormal gene regulation (such as promoter methylation, down-regulation of transcription factors PAX8 / NKX2-1 expression), abnormal signal pathways (continuous activation of the BRAF V600E mutation inhibits NIS transcription by the MAPK / ERK pathway), epigenetic modification (histone deacetylase HDACs inhibit NIS gene transcription, miR-146a / 221 / 222 target NIS mRNA), and inhibition of NIS expression by Snail / Twist during the epithelial-mesenchymal transition (EMT) process. This change causes cancer cells to lose the ability to take up iodine, making RAI ineffective, and is accompanied by the activation of the MAPK / PI3K pathway to promote tumor malignant progression, ultimately leading to poor prognosis of patients. At present, studies aim to restore NIS expression to improve the treatment effect of IRTC through strategies such as epigenetic drugs (decitabine reverses methylation, vorinostat inhibits HDACs), targeted inhibitors (vemurafenib combined with trametinib blocks the BRAF / MEK pathway), miRNA antagonists, and adenovirus-mediated NIS gene delivery.

[0004] Dioscoreae bulbiferae Rhizoma, also known as Dioscorea bulbifera L., is the dried tuber of Dioscorea bulbifera L. of the genus Dioscorea in the Dioscoreaceae family. It has the effects of detoxifying and detumescence, resolving phlegm and dissipating nodules, cooling blood and stopping bleeding. It is commonly used for thyroid nodules, scrofula, sore throat, hematemesis, hemoptysis, pertussis, tumors, gastric cancer, colorectal cancer, cervical cancer, AIDS, bacterial infections, etc. It has a unique effect on thyroid tumors and thyroid adenomas. There is no study publicly reporting its therapeutic effect on radioiodine-refractory differentiated thyroid cancer.

[0005] Among papillary thyroid carcinoma (PTC), radioactive iodinerefractory DTC (RAIR-DTC) is the main factor affecting the prognosis of PTC and is a difficult point in clinical treatment, and new treatment options are needed. Currently, there is a lack of research on the treatment of this type of thyroid cancer with Chinese medicine prescriptions and drugs, and there is no research on the mechanism by which iodine-refractory DTC can reverse the iodine uptake effect. Summary of the invention

[0006] The present invention provides a Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer, a preparation method and application thereof, so as to reverse the iodine uptake effect of iodine-refractory differentiated thyroid cancer, promote the death of cancer cells through the ferroptosis pathway, and improve the anti-tumor activity.

[0007] In order to solve the above technical problems, one of the purposes of the present invention is to provide a Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer, which is prepared by solvent extraction of Chinese medicine raw materials, and the Chinese medicine raw materials include white peony root, roasted licorice, Dioscorea bulbifera, Astragalus and stir-fried Atractylodes.

[0008] In the Chinese medicine formula of the present application, Dioscorea bulbifera has the effects of detoxification and swelling, phlegm-resolving and stagnation-dispersing, cooling blood and stopping bleeding, and has its unique effect in anti-thyroid tumor. Astragalus has the effects of strengthening the spleen and replenishing the middle, raising yang and lifting the sunken, benefiting the defense and consolidating the exterior, diuresis, toxin-regenerating and anti-tumor. White peony root contains various chemical components such as paeoniflorin, which can inhibit tumor cell proliferation and induce tumor cell apoptosis. It may enhance the recognition and killing ability of immune cells to tumor cells by regulating the immune function of the body, and may also participate in regulating some signal transduction pathways in tumor cells to hinder the abnormal growth and spread of tumor cells. Atractylodes macrocephala contains ingredients such as volatile oil and atractylodes ketone. Atractylodes macrocephala may indirectly affect the occurrence and development of tumors by regulating the internal environment homeostasis of the body, enhancing the body's antioxidant capacity, regulating immune response, and improving intestinal microecology. Licorice contains many ingredients such as glycyrrhizic acid and glycyrrhetinic acid. On the one hand, it has anti-inflammatory effects. Chronic inflammation is closely related to the occurrence and development of tumors. It can indirectly fight tumors by alleviating the inflammatory microenvironment. On the other hand, licorice can also regulate the body's immune regulatory network, stimulate the activity of immune cells, and assist the immune system to better monitor and eliminate tumor cells. The Chinese medicine compatibility ingredients of this application are matched through various Chinese medicine components, and the ingredients are suitable, which helps to enhance the efficacy, can reverse the iodine uptake of iodine-refractory differentiated thyroid cancer, and have an anti-tumor effect, promoting the death of tumor cells through the ferroptosis pathway.

[0009] As a preferred embodiment, the Chinese medicinal raw materials include the following components in parts by weight: 15-25 parts of white peony root, 5-15 parts of roasted liquorice root, 10-20 parts of Dioscorea bulbifera, 40-60 parts of Astragalus and 20-40 parts of stir-fried Atractylodes.

[0010] Preferably, the traditional Chinese medicine raw materials include the following components in parts by weight: 18 - 22 parts of white peony root, 8 - 12 parts of honey - fried licorice root, 13 - 18 parts of dioscorea bulbifera, 45 - 55 parts of astragalus membranaceus, and 25 - 35 parts of stir - fried atractylodes macrocephala.

[0011] Preferably, the traditional Chinese medicine raw materials include the following components in parts by weight: 20 parts of white peony root, 10 parts of honey - fried licorice root, 15 parts of dioscorea bulbifera, 50 parts of astragalus membranaceus, and 30 parts of stir - fried atractylodes macrocephala.

[0012] Preferably, the solvent is water.

[0013] Preferably, the mass ratio of the traditional Chinese medicine raw materials to the solvent is 1:(0.2 - 5).

[0014] Preferably, the mass ratio of the traditional Chinese medicine raw materials to the solvent is 1:(1 - 3).

[0015] To solve the above - mentioned technical problems, the second object of the present invention is to provide a preparation method of an extract of a traditional Chinese medicine compatibility formula for treating iodine - refractory thyroid cancer, comprising the following steps:

[0016] (1) Accurately weigh the traditional Chinese medicine raw materials of white peony root, honey - fried licorice root, dioscorea bulbifera, astragalus membranaceus, and stir - fried atractylodes macrocephala, put them into a decocting utensil, add half of the solvent, soak, then heat and decoct, and filter to obtain the first decoction.

[0017] (2) Add the remaining solvent to the medicinal residues, repeat the decocting process in step (1), filter to obtain the second decoction, combine the two decoctions, and concentrate to obtain the extract.

[0018] Preferably, in steps (1) and (2), the soaking time is 10 - 60 min, boil with strong fire, and then turn to slow fire and decoct for 20 - 60 min.

[0019] Preferably, in step (2), the decoction is concentrated to 5wt% - 30wt% of the total volume of the added solvent.

[0020] To solve the above - mentioned technical problems, the third object of the present invention is to provide an application of an extract of a traditional Chinese medicine compatibility formula for treating iodine - refractory thyroid cancer in the preparation of a drug for treating iodine - refractory thyroid cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. In the past, traditional Chinese medicine compatibility only had an inhibitory effect on thyroid tumors, and there has been no research on its effect on iodine-refractory differentiated thyroid cancer. The extract of the traditional Chinese medicine compatibility formula provided by the present invention can reverse the iodine uptake of iodine-refractory differentiated thyroid cancer and has an inhibitory effect on its malignant biological behavior. By promoting the inhibition of GPX4 expression to promote ferroptosis, the anti-tumor activity is thus enhanced.

[0023] 2. The extract of the traditional Chinese medicine compatibility formula in this application has the effects of cooling blood and reducing fire, dissipating stasis and detoxifying, resolving phlegm and dissipating nodules, has a relatively mild degree of damage to the liver, and has small side effects. After the compatibility of the traditional Chinese medicine components, it can also reduce the hepatotoxicity of Dioscoreae bulbiferae and enhance the anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : It is the NIS protein expression of tumors in mice detected by western blot method after drug intervention in Experimental Example 3 of the present invention (Note: Experimental Group 1 - Example 1; Experimental Group 2 - Comparative Example 1; Experimental Group 3 - Comparative Example 2; Experimental Group 4 - Comparative Example 4);

[0025] Figure 2 : It is the protein expression of ferroptosis-related genes in tumors in mice detected by western blot method after drug intervention in Experimental Example 3 of the present invention (Note: Experimental Group 1 - Example 1; Experimental Group 2 - Comparative Example 1; Experimental Group 3 - Comparative Example 2; Experimental Group 4 - Comparative Example 4). DETAILED DESCRIPTION OF THE INVENTION

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded within the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Example 1

[0030] An extract of a traditional Chinese medicine compatibility formula for treating iodine-refractory thyroid cancer, which is obtained by extracting traditional Chinese medicine raw materials with water. The traditional Chinese medicine raw materials include 20 g of white peony root, 10 g of roasted licorice root, 15 g of Dioscoreae bulbiferae Rhizoma, 50 g of astragalus membranaceus, and 30 g of stir-fried atractylodes macrocephala. Its preparation method includes the following steps:

[0031] (1) Accurately weigh the traditional Chinese medicine raw materials of white peony root, roasted licorice root, Dioscoreae bulbiferae Rhizoma, astragalus membranaceus, and stir-fried atractylodes macrocephala according to the formula, put them into a decocting utensil, add 250 mL of water, soak for 30 min first, then boil with strong fire, and then turn to slow fire and decoct for 30 min, filter to obtain the first decoction;

[0032] (2) Add 250 ml of water to the medicinal residues again, repeat the decocting process in step (1) to obtain the second decoction, combine the two decoctions, concentrate to 10 wt% of the total volume of the added water, and store at 4°C for later use.

[0033] Comparative Example 1

[0034] An extract of a traditional Chinese medicine compatibility formula for treating iodine-refractory thyroid cancer, which is obtained by extracting traditional Chinese medicine raw materials with water. The traditional Chinese medicine raw materials include 30 g of white peony root and 20 g of roasted licorice root. Its preparation method includes the following steps:

[0035] (1) Accurately weigh the traditional Chinese medicine raw materials of white peony root and roasted licorice root according to the formula, put them into a decocting utensil, add 250 mL of water, soak for 30 min first, then boil with strong fire, and then turn to slow fire and decoct for 30 min, filter to obtain the first decoction;

[0036] (2) Add 250 ml of water to the medicinal residues again, repeat the decocting process in step (1) to obtain the second decoction, combine the two decoctions, concentrate to 10 wt% of the total volume of the added water, and store at 4°C for later use.

[0037] Comparative Example 2

[0038] An extract of a traditional Chinese medicine compatibility formula for treating iodine-refractory thyroid cancer, which is obtained by extracting traditional Chinese medicine raw materials with water. The traditional Chinese medicine raw materials include 30 g of atractylodes macrocephala and 10 g of roasted licorice root. Its preparation method includes the following steps:

[0039] (1) Weigh the traditional Chinese medicine raw materials of Atractylodes macrocephala and roasted licorice accurately according to the formula, put them into the decocting appliance, add 250 mL of water, soak for 30 min first, then boil with strong fire, and then turn to slow fire and decoct for 30 min, filter to obtain the first decoction.

[0040] (2) Add another 250 ml of water to the medicinal residues, repeat the decocting process in step (1) to obtain the second decoction, combine the two decoctions, concentrate to 10 wt% of the total volume of the added water, and store at 4 °C for later use.

[0041] Control Example 3

[0042] An extract of a traditional Chinese medicine compatibility formula for treating iodine-refractory thyroid cancer, which is obtained by extracting traditional Chinese medicine raw materials with water. The traditional Chinese medicine raw materials include 50 g of Astragalus membranaceus and 10 g of roasted licorice. Its preparation method includes the following steps:

[0043] (1) Weigh the traditional Chinese medicine raw materials of Astragalus membranaceus and roasted licorice accurately according to the formula, put them into the decocting appliance, add 250 mL of water, soak for 30 min first, then boil with strong fire, and then turn to slow fire and decoct for 30 min, filter to obtain the first decoction.

[0044] (2) Add another 250 ml of water to the medicinal residues, repeat the decocting process in step (1) to obtain the second decoction, combine the two decoctions, concentrate to 10 wt% of the total volume of the added water, and store at 4 °C for later use.

[0045] Experimental Example 1

[0046] Determination of the proliferation rate of thyroid cancer GLAG-66 and IHH4 cell lines by CCK-8 method

[0047] Cell source and culture:

[0048] 1) The IHH-4 and GLAG-66 cell lines are obtained from the supplier and cultured in RPMI-1640 medium containing fetal bovine serum, penicillin and streptomycin, and placed in an incubator at 37 °C and 5% CO2. When the cell confluence reaches about 80%-90%, subculture is carried out. First, discard the old medium in the culture flask, gently rinse the cells twice with phosphate buffer solution to remove the residual medium and serum. Then, add an appropriate amount of 0.25% trypsin-EDTA solution to cover the cell surface, place the culture flask in an incubator at 37 °C for digestion for about 3 min. When the cells become round and the gaps increase under the microscope, add an equal volume of serum-containing medium to terminate digestion. Gently pipette the cells to form a single-cell suspension, subculture according to a ratio of 1:4, inoculate into a new culture flask, and continue to culture.

[0049] 2) Animal selection and grouping: The traditional Chinese medicine compatibility extract prepared in Example 1 was used as experimental group 1, the traditional Chinese medicine compatibility extracts prepared in Comparative Examples 1-3 were used as experimental groups 2-4, an equal amount of normal saline was used as the blank control group, and vemurafenib was used as the positive control group; healthy SD rats with a body weight of 200-250 g were selected. After 3 days of adaptive feeding, they were randomly divided into 5 groups, with 3 rats in each group, corresponding to the experimental group, the control group, and the blank control group respectively.

[0050] 3) Drug administration and collection of drug-containing serum: The animals in the experimental group and the positive control group were given the corresponding drugs by gavage at a dose of 10 ml / kg body weight, the blank control group was given an equal amount of normal saline, and the drugs were administered 2 times a day for 7 consecutive days. 1 h after the last drug administration, the animals were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta. The blood was collected in a sterile centrifuge tube, allowed to stand at room temperature for 2 h, and then centrifuged at 3000 rpm for 15 min to separate the serum. The serum was filtered and sterilized through a 0.22 μm filter membrane, the complement was inactivated in a water bath at 56 °C for 30 min, and after being aliquoted, it was stored at -20 °C for later use.

[0051] 4) Intervention of drug-containing serum on cells: The cells were inoculated into 96-well plate culture vessels at an appropriate density of 5×10 3 cells per well, cultured for 24 h to allow the cells to adhere to the wall, the old culture medium was discarded, and fresh culture medium containing the drug-containing serum of each group was added respectively. The final concentration of the drug-containing serum was set at 10%, and the cells were cultured for another 48 h, and then subsequent cell proliferation and related protein expression were detected.

[0052] 5) Detection of cell proliferation (CCK-8 method): GLAG-66 or IHH4 cells were inoculated. The cells in the logarithmic growth phase were digested with trypsin to make a single-cell suspension and counted. The cells were inoculated into 96-well plates, with a volume of 100 μL per well. Each group was set with 5 replicates, and the edge wells were filled with sterile PBS to reduce the edge effect. The 96-well plates were placed in an incubator at 37 °C and 5% CO2 for 24 h to allow the cells to adhere to the wall. Drug treatment: The old culture medium in the 96-well plates was discarded, and according to the above grouping, fresh culture medium containing the drug-containing serum of each group was added respectively. The final concentration of the drug-containing serum was set at 10%, 100 μL per well. The cells were cultured for another 24 h, 48 h, and 72 h. Detection of cell proliferation: Before detection at each time point, 10 μL of CCK-8 solution was added to each well, gently mixed, and the 96-well plates were placed in an incubator at 37 °C and 5% CO2 for 3 h. Then, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. The formula for calculating the cell proliferation inhibition rate is: inhibition rate = (1 - OD value of the experimental group / OD value of the blank control group) × 100%.

[0053] Table 1 - Results of the proliferation inhibition rate of each group of drugs on thyroid cancer cell lines detected by the CCK-8 method

[0054]

[0055]

[0056] The experimental results are shown in Table 1. The extract after decoction of the compatibility formula of Example 1, which consists of Paeonia lactiflora Pall., Radix Glycyrrhizae Preparata, Dioscoreae bulbiferae Rhizoma, Astragalus membranaceus (Fisch.) Bunge, and Atractylodes macrocephala Koidz. stir-fried with bran, has a good inhibitory effect on the proliferation of human papillary thyroid carcinoma cells. The proliferation inhibition rate reaches 74%, even higher than that of the positive reference vemurafenib, indicating that the extract has anti-tumor activity. The proliferation inhibition rates of the extracts of the compatibility components in Comparative Examples 1-3 are reduced by 30%-44% compared with that of Example 1, and the sensitivity and inhibitory effect on thyroid carcinoma cells are significantly inferior to those of the extract of Example 1, indicating insufficient therapeutic effect.

[0057] Test Example 2

[0058] Evaluation of anti-tumor activity by nude mouse tumorigenesis experiment

[0059] 1) Cell preparation: Select a suitable tumor cell line (GLAG-66 cells) and culture it in a cell incubator until the logarithmic growth phase. Digest the cells with trypsin to make a single-cell suspension, count the number of cells, and adjust the cell concentration to 5×10 6 cells / mL.

[0060] 2) Preparation and grouping of nude mice: Select SPF-grade nude mice at 4-6 weeks of age with a body weight of 18-22 g and adaptively raise them for 5 days. Randomly divide the nude mice into 4 groups with 3 mice in each group. The traditional Chinese medicine compatibility extract prepared in Example 1 is used as experimental group 1, and the traditional Chinese medicine compatibility extracts prepared in Comparative Examples 1-3 are used as experimental groups 2-4.

[0061] 3) Inoculation of tumor cells: Use a 1 mL syringe to aspirate an appropriate amount of cell suspension (1×10 6 cells are inoculated into each nude mouse), and inoculate the tumor cells subcutaneously in the right axilla of the nude mouse. When inoculating, gently pinch the skin of the nude mouse, insert the needle into the subcutaneous tissue, and slowly inject the cell suspension to avoid leakage. After inoculation, observe the general state of the nude mouse and the situation of the inoculation site every day.

[0062] 4) Administration dose and method: When the tumor grows to a certain volume and the tumor diameter is about 6 mm, start administering the drug. The nude mice in the experimental groups are intragastrically administered the corresponding extracts at a dose of 10 ml / kg body weight respectively, and the blank control group is administered an equal amount of normal saline once a day for 21 consecutive days.

[0063] 5) Measurement of tumor volume: Measure the long diameter (a) and short diameter (b) of the tumor with a vernier caliper every 3 days, and according to the formula V = 1 / 2×a×b 2Calculate the tumor volume. After the intervention ended, the tumor was removed and weighed. The data are shown in Table 2 below. The results indicate that the tumor weight of Experimental Group 1 was the smallest after the intervention, suggesting that the anti-tumor effect of the traditional Chinese medicine compatibility extract in Example 1 was the best.

[0064] Table 2 - Tumor weights of mice in the nude mouse tumorigenesis experiment 21 days after drug administration

[0065]

[0066] As shown in Table 2, the extract of the formula components in Experimental Group 1 had the strongest inhibitory effect on the growth of mouse tumors, the lowest mouse tumor weight, and the best anti-tumor effect. The extract of the formula components in Experimental Group 2 had the weakest inhibitory effect on the growth of mouse tumors, the heaviest mouse tumor weight, and the worst anti-tumor effect. Although the extracts of the formula components in Experimental Groups 3 - 4 had a certain anti-tumor effect compared with the blank control group, the treatment effect was not as good as that of Experimental Group 1.

[0067] Test Example 3

[0068] Detection of the expression of the important index NIS protein and ferroptosis-related gene proteins for the iodine uptake rate in iodine-refractory thyroid cancer by western blot method

[0069] After the intervention in the nude mouse tumorigenesis experiment of Test Example 2 ended, the mice were anesthetized, and then the tumor bodies were surgically removed. Total proteins of the tumor bodies were extracted, and steps such as protein quantification, SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and chemiluminescence detection were carried out to analyze the expression changes of related proteins. The steps are as follows:

[0070] 1) Tumor body treatment and collection: Extract total proteins of the tumor body, measure the protein concentration using a BCA protein quantification kit, operate according to the kit instructions, draw a standard curve, and calculate the protein concentration of the sample.

[0071] 2) SDS-PAGE electrophoresis: According to the protein quantification results, take an appropriate amount of protein sample, add 5×Loading Buffer, and denature in a boiling water bath for 5 minutes. Load the denatured protein sample onto the SDS-PAGE gel and perform electrophoresis. The electrophoresis conditions are: stacking gel 80V, separating gel 120V until the bromophenol blue indicator reaches the bottom of the gel.

[0072] 3) Membrane transfer: Transfer the protein from the gel to a PVDF membrane (pore size: 0.45μm) using the wet transfer method. The membrane transfer conditions are: 200mA, constant current, and the membrane transfer time is adjusted according to the molecular weight of the target protein.

[0073] 4) Blocking: Put the transferred PVDF membrane into 5% skim milk powder (prepared with TBST, TBST: Tris-Buffered Saline with Tween20) and block at room temperature for 1 hour.

[0074] 5) Primary antibody incubation: Place the blocked PVDF membrane into the primary antibody dilution solution (prepared with 5% skim milk powder) and incubate overnight at 4°C.

[0075] 6) Membrane washing: Wash the membrane 3 times with TBST solution for 10 minutes each time to remove unbound primary antibody.

[0076] 7) Secondary antibody incubation: Place the PVDF membrane into the corresponding HRP-labeled secondary antibody dilution solution (prepared with 5% skim milk powder and the secondary antibody dilution ratio is 1:5000), and incubate at room temperature for 1 hour.

[0077] 8) Membrane washing: Wash the membrane again 3 times with TBST solution for 10 minutes each time to remove unbound secondary antibody.

[0078] 9) Chemiluminescence detection: Place the PVDF membrane into the chemiluminescence substrate, operate according to the instructions, make the substrate bind to the HRP on the membrane, then place the membrane into the chemiluminescence imaging system for exposure, collect images, and use ImageJ software to analyze the gray value of the protein bands.

[0079] The experimental results are as Figure 1 shown, which is the expression of NIS protein in tumors after the nude mouse tumorigenesis test. NIS protein is an important indicator reflecting the iodine uptake rate of iodine-refractory thyroid cancer. The higher the expression of NIS protein, the better the effect of reversing the iodine uptake rate. The expression level of NIS protein in tumors in experimental group 1 is the highest, indicating that the traditional Chinese medicine compatibility extract in Example 1 has the best therapeutic effect on iodine-refractory thyroid cancer.

[0080] The experimental results are as Figure 2 shown, which is the expression of ferroptosis-related gene proteins in tumors after the nude mouse tumorigenesis test. The expression of GPX4 in tumors in experimental group 1 was significantly inhibited. Ferroptosis is a newly discovered form of iron-dependent cell death. The applicant's experimental research shows that there is overexpression of the key ferroptosis target GPX4 in iodine-refractory differentiated thyroid cancer. Adding ferroptosis inducers can inhibit its malignant biological behavior. Therefore, inhibiting the expression of GPX4 can promote ferroptosis in thyroid cancer, thus having anti-tumor activity.

[0081] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. In particular, it is pointed out that for those skilled in the art, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer, characterized in that: The medicine is prepared by solvent extraction from Chinese medicinal raw materials, wherein the Chinese medicinal raw materials include white peony root, roasted liquorice root, Dioscorea bulbifera, Astragalus and stir-fried Atractylodes macrocephala.

2. The Chinese medicinal formula extract for treating iodine-refractory thyroid cancer according to claim 1, characterized in that: The Chinese medicinal raw materials include the following components by weight: 15-25 parts of white peony root, 5-15 parts of roasted liquorice root, 10-20 parts of Dioscorea bulbifera, 40-60 parts of Astragalus and 20-40 parts of stir-fried Atractylodes macrocephala.

3. The Chinese medicinal formula extract for treating iodine-refractory thyroid cancer according to claim 1, characterized in that: The Chinese medicinal raw materials include the following components in parts by weight: 18-22 parts of white peony root, 8-12 parts of roasted liquorice root, 13-18 parts of Dioscorea bulbifera, 45-55 parts of Astragalus and 25-35 parts of stir-fried Atractylodes macrocephala.

4. The Chinese medicinal formula extract for treating iodine-refractory thyroid cancer according to claim 1, characterized in that: The Chinese medicinal raw materials include the following components by weight: 20 parts of white peony root, 10 parts of roasted liquorice root, 15 parts of Dioscorea bulbifera, 50 parts of Astragalus and 30 parts of stir-fried Atractylodes macrocephala.

5. The Chinese medicinal formula extract for treating iodine-refractory thyroid cancer according to claim 1, characterized in that: The solvent is water.

6. The Chinese medicinal formula extract for treating iodine-refractory thyroid cancer according to claim 1, characterized in that: The mass ratio of the Chinese medicinal raw material to the solvent is 1:(0.2-5).

7. A method for preparing a Chinese medicine compatibility formula extract for treating iodine-refractory thyroid cancer as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Accurately weigh the raw materials of white peony root, roasted liquorice root, Dioscorea bulbifera, Astragalus membranaceus and stir-fried Atractylodes macrocephala, put them into a decoction vessel, add half of the solvent to soak, heat and decoct, filter, and obtain the first medicinal solution; (2) adding the remaining solvent to the medicinal residue, repeating the decoction process of step (1), filtering to obtain a second medicinal solution, combining the two medicinal solutions, and concentrating to obtain an extract.

8. The method for preparing the Chinese medicinal compatibility formula extract for treating iodine-refractory thyroid cancer according to claim 7, characterized in that: In steps (1) and (2), the soaking time is 10-60 minutes, the mixture is boiled over high heat, and then simmered over low heat for 20-60 minutes.

9. The method for preparing the Chinese medicinal compatibility formula extract for treating iodine-refractory thyroid cancer according to claim 7, characterized in that: In step (2), the drug solution is concentrated to 5wt%-30wt% of the total volume of the added solvent.

10. Use of the Chinese medicinal formula extract for treating iodine-refractory thyroid cancer as claimed in any one of claims 1 to 6 in the preparation of a drug for treating iodine-refractory thyroid cancer.

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