A Chinese medicine monomer for targeted degradation of PTPN22 protein and its application

Abstract

The invention discloses a Chinese medicine monomer agent for targeted degradation of PTPN22 protein and its application, belongs to the field of medical technology, and specifically relates to a Chinese medicine monomer agent, including: a Chinese medicine monomer and an adjuvant; the Chinese medicine monomer includes at least one of paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin, the adjuvant includes at least one of a medical solvent, and the medical solvent includes at least one of DMSO, ethanol, isopropanol, acetone, benzyl alcohol, ethyl acetate, polyoxyethylene castor oil and water; the amount of the Chinese medicine monomer used is 0.001-0.05wt% of the medical solvent. The invention also discloses the use of the above-mentioned Chinese medicine monomer agent in preparing a compound for degrading PTPN22 protein and / or a compound for increasing the expression of NIS protein and / or a compound for increasing the iodine uptake capacity of cells and / or a drug for treating thyroid cancer and / or a drug for treating iodine-resistant thyroid cancer and / or a drug for treating diseases caused by abnormal expression of PTPN22 protein.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a Chinese medicine monomer agent for targeted degradation of PTPN22 protein and application thereof. Background Art

[0002] Thyroid cancer is one of the most common malignancies in the endocrine system, and iodine-131 radiotherapy is often used in its treatment. However, many thyroid cancer cells show resistance to iodine, resulting in poor treatment efficacy. Iodine resistance is closely related to the downregulation of the expression of sodium-iodide symporter (NIS), which is a key protein for iodine uptake by thyroid cells. In thyroid cancer cells, the expression of NIS is regulated by multiple factors.

[0003] PTPN22, or protein tyrosine phosphatase non-receptor type 22, is a phosphatase associated with a variety of autoimmune diseases. There is currently a lack of effective PTPN22 degraders to upregulate the expression of NIS to reverse the iodine resistance of thyroid cancer. Existing research mainly focuses on the development of PTPN22 inhibitors, but these inhibitors often fail to specifically degrade PTPN22 protein and may be accompanied by nonspecific binding and side effects. In addition, inhibitors obtained by traditional chemical synthesis methods may have problems such as low bioavailability and poor stability, which limits their application in clinical treatment. PTPN22 protein is a phosphatase associated with a variety of autoimmune diseases, and its abnormal expression may lead to the development of the disease. Traditional small molecule inhibitors are difficult to achieve effective degradation of PTPN22 protein. Therefore, the development of new degraders is of great significance for the treatment of related diseases.

[0004] The present invention aims to provide a new strategy to reverse the iodine resistance of thyroid cancer by upregulating the expression of NIS through specific degradation of PTPN22 protein. We found that benzoylpaeoniflorin, an active ingredient extracted from the traditional Chinese medicine peony, can effectively degrade PTPN22 protein and show an upregulation effect on NIS expression in an in vitro model, providing a new perspective for the treatment of thyroid cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a Chinese medicine monomer agent for targeted degradation of PTPN22 protein and its application, which can target and degrade PTPN22 protein, increase the expression of NIS protein, improve the iodine uptake capacity, treat iodine-resistant thyroid cancer drugs, and treat diseases caused by abnormal expression of PTPN22 protein.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] A Chinese medicine monomer agent comprises: a Chinese medicine monomer and an adjuvant; the Chinese medicine monomer comprises at least one of paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin, the adjuvant comprises at least a medical solvent, and the medical solvent comprises at least one of DMSO, ethanol, isopropanol, acetone, benzyl alcohol, ethyl acetate, polyoxyethylene castor oil and water; the amount of the Chinese medicine monomer used is 0.001-0.05wt% of the medical solvent. The present invention studies Chinese medicine monomers including paeoniflorin, paeoniflorin lactone glycosides, benzoylpeoniflorin and hydroxypeoniflorin, and finds that paeoniflorin lactone glycosides have poor effects, while paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin all have good effects, can target and degrade PTPN22 protein, increase the expression of NIS protein, increase the iodine uptake capacity, treat iodine-resistant thyroid cancer and treat abnormal expression of PTPN22 protein.

[0008] Preferably, the adjuvant further comprises an auxiliary agent, and the auxiliary agent comprises an amino alcohol benzoate compound, and the amino alcohol benzoate compound has an ethyl benzoate group and an ethyl aminoacetate group. The present invention also combines 3-hydroxybenzoic acid ethyl ester, epichlorohydrin and ethyl 2-aminoacetate to form an amino alcohol benzoate compound. By compounding the amino alcohol benzoate compound with a traditional Chinese medicine monomer, the effect of targeted degradation of PTPN22 protein, the effect of increasing the expression of NIS protein, the effect of increasing the iodine uptake ability of cells, the treatment of iodine-resistant thyroid cancer and the treatment of abnormal expression of PTPN22 protein can be improved.

[0009] Preferably, the aminobenzoate compound is prepared by combining ethyl 3-hydroxybenzoate, epichlorohydrin and ethyl 2-aminoacetate.

[0010] Preferably, the amount of epichlorohydrin used is 40-60wt% of ethyl 3-hydroxybenzoate, and the amount of ethyl 2-aminoacetate used is 60-120wt% of ethyl 3-hydroxybenzoate.

[0011] Preferably, the amino alcohol benzoate compound is used in an amount of 0.001-0.01 wt % of the medicinal solvent.

[0012] Preferably, the traditional Chinese medicine monomer includes any one of paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin.

[0013] Preferably, the Chinese medicine monomer includes any two of paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin. If more than one Chinese medicine monomer is used, the Chinese medicine monomers can be mixed in any proportion.

[0014] Preferably, the Chinese medicine monomers include paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin. If more than one Chinese medicine monomer is used, the Chinese medicine monomers can be mixed in any proportion.

[0015] The invention discloses use of a Chinese medicine monomer agent in preparing a compound for degrading PTPN22 protein and / or a compound for increasing the expression of NIS protein and / or a compound for increasing the iodine uptake capacity of cells and / or a drug for treating thyroid cancer and / or a drug for treating iodine-resistant thyroid cancer and / or a drug for treating diseases caused by abnormal expression of PTPN22 protein; the Chinese medicine monomer agent contains a Chinese medicine monomer, which includes at least one of paeoniflorin, benzoylpeoniflorin and hydroxypeoniflorin; the Chinese medicine monomer agent contains a medical solvent, and the use amount of the Chinese medicine monomer is 0.001-0.05wt% of the medical solvent.

[0016] Preferably, the medical solvent is ethanol; or, the medical solvent is ethanol and polyoxyethylated castor oil; or, the medical solvent is isopropanol; or, the medical solvent is ethyl acetate.

[0017] Preferably, the Chinese medicine monomer also contains an amino alcohol benzoate compound.

[0018] Preferably, the ethanol and polyoxyethylated castor oil in the medical solvent are mixed in a mass ratio of 1:0.01-0.3.

[0019] Preferably, in the preparation of the amino alcohol benzoate compound, ethyl 3-hydroxybenzoate, anhydrous potassium carbonate and potassium iodide are added to DMF and stirred, epichlorohydrin is added at 20-40°C, the pH is adjusted to 9-10, and then the reaction is carried out at 120-180°C for 3-12 hours. After the reaction is completed, the intermediate product is separated by column chromatography, the intermediate product is mixed with ethanol, the amino alcohol compound is added, the reaction is carried out at 70-90°C for 3-12 hours, and water is added to precipitate to obtain the amino alcohol benzoate compound.

[0020] More preferably, in the preparation of the amino alcohol benzoate compound, the amount of ethyl 3-hydroxybenzoate used is 5-20wt% of DMF.

[0021] More preferably, in the preparation of the amino alcohol benzoate compound, the amount of anhydrous potassium carbonate used is 30-60wt% of ethyl 3-hydroxybenzoate.

[0022] More preferably, in the preparation of the amino alcohol benzoate compound, the amount of potassium iodide used is 20-50 wt % of ethyl 3-hydroxybenzoate.

[0023] More preferably, in the preparation of the amino alcohol benzoate compound, the amount of epichlorohydrin used is 40-60wt% of ethyl 3-hydroxybenzoate.

[0024] More preferably, in the preparation of the aminoalcohol benzoate compound, the amount of the intermediate product is measured as ethyl 3-hydroxybenzoate, the aminoalcohol compound is ethyl 2-aminoacetate, and the amount of the aminoalcohol compound is 60-120wt% of ethyl 3-hydroxybenzoate.

[0025] The invention discloses a Chinese medicine monomer agent, which comprises a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, and the adjuvant comprises a medical solvent and / or an auxiliary agent.

[0026] Preferably, the medical solvent is any one or more of DMSO, ethanol, isopropanol, acetone, benzyl alcohol, ethyl acetate, polyoxyethylated castor oil and water.

[0027] Preferably, the medical solvent is ethanol and polyoxyethylated castor oil, and the ethanol and polyoxyethylated castor oil in the medical solvent are mixed in a mass ratio of 1:0.01-0.3.

[0028] Preferably, the medical solvent is water and isopropanol, and the water and isopropanol in the medical solvent are mixed in a mass ratio of 1:0.01-0.3.

[0029] Preferably, the amount of benzoylpeonidin used is 0.001-0.05wt% of the medical solvent.

[0030] Preferably, the auxiliary agent comprises an amino alcohol benzoate compound, and the usage amount of the amino alcohol benzoate compound is 0.001-0.01wt% of the medical solvent.

[0031] Preferably, the auxiliary agent also includes 1-O-p-coumaryl glycerol. The amount of 1-O-p-coumaryl glycerol used is 0.0001-0.003wt% of the medical solvent. When the present invention uses the amino alcohol benzoate compound and the traditional Chinese medicine monomer, 1-O-p-coumaryl glycerol can also be added. When the amino alcohol benzoate compound is used together with the traditional Chinese medicine monomer and 1-O-p-coumaryl glycerol, the effect of targeted degradation of PTPN22 protein can be further improved, the effect of increasing the expression of NIS protein, the effect of increasing the iodine uptake ability of cells, the treatment of iodine-resistant thyroid cancer and the treatment of abnormal expression of PTPN22 protein can be further improved.

[0032] The present invention discloses that benzoylpaeoniflorin can effectively target and degrade PTPN22 protein, and benzoylpaeoniflorin can be used to treat and reverse iodine-resistant thyroid cancer, thereby reversing iodine resistance by increasing the sensitivity of thyroid cancer cells to iodine.

[0033] The benzoylpeonidin disclosed in the present invention is a natural compound extracted from traditional Chinese medicine, which has low toxicity and good biocompatibility, which makes it safer in clinical applications. By specifically degrading PTPN22 protein, the benzoylpeonidin derivative can reduce the impact on normal cells and reduce side effects. The compound provided by the present invention can increase the sensitivity of thyroid cancer cells to iodine, providing a new strategy for the treatment of iodine-resistant thyroid cancer.

[0034] The present invention adopts Chinese medicine monomers extracted from Chinese medicine and is prepared with auxiliary agents to form Chinese medicine monomer agents, wherein the auxiliary agents include medical solvents and auxiliary agents, wherein the medical solvents include at least one of DMSO, ethanol, isopropanol, acetone, benzyl alcohol, ethyl acetate, polyoxyethylene castor oil and water, and the auxiliary agent can be selected from amino alcohol benzoate compounds formed by combining 3-hydroxybenzoic acid ethyl ester, epichlorohydrin and 2-aminoethyl acetate, thus having the following beneficial effects: targeted degradation of PTPN22 protein, improvement of NIS protein expression, improvement of iodine uptake capacity, treatment of iodine-resistant thyroid cancer and treatment of abnormal expression of PTPN22 protein. Therefore, the present invention is a Chinese medicine monomer agent and its application for targeted degradation of PTPN22 protein that can target degradation of PTPN22 protein, improvement of NIS protein expression, improvement of iodine uptake capacity, treatment of iodine-resistant thyroid cancer drugs and treatment of diseases caused by abnormal expression of PTPN22 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram showing the results of screening differentiated thyroid cancer cell lines expressing PTPN22.

[0036] Figure 2 This is the result diagram of the molecular docking model based on PTPN22.

[0037] Figure 3 This is a graph showing the results of the thermal stability experiment of PTPN22 protein.

[0038] Figure 4 This is a graph showing the results of binding affinity testing.

[0039] Figure 5 Benzoylpaeoniflorin regulates the expression of PTPN22 protein.

[0040] Figure 6 This figure shows the effect of ribosome inhibitors on benzoylpeonidin-induced PTPN22 protein degradation.

[0041] Figure 7 This is a graph showing the results of PTPN22 protein half-life detection.

[0042] Figure 8 The result diagram of Co-IP verification of benzoylpaeoniflorin ubiquitination and degradation of PTPN22 protein.

[0043] Fig. 9 This is a graph showing the effect of benzoylpaeoniflorin on total cell protein expression.

[0044] Fig.10 This is a graph showing the effect of benzoylpaeoniflorin on the expression of cell membrane proteins.

[0045] Fig.11This is a graph showing the effect of benzoylpeonidin on iodine uptake function.

[0046] Fig.12 This is a molecular docking model diagram.

[0047] Fig.13 This is a graph showing the results of co-immunoprecipitation experiments.

[0048] Fig.14 To verify the expression level of NIS protein after knockdown / overexpression of PTPN22.

[0049] Fig.15 The relative expression of NIS protein.

[0050] Fig.16 This is a graph of iodide ion content. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0053] The research basis of the present invention is as follows:

[0054] (1) Screening of differentiated thyroid cancer cell lines expressing PTPN22

[0055] The protein expression of thyroid cancer cell lines and normal thyroid cells Nthy-ori 3-1 was studied, and the expression level of PTPN22 protein was quantitatively analyzed by Western Blot technology. By extracting total protein from multiple thyroid cancer cell lines and normal rat and mouse thyroid cells, the present invention successfully screened out differentiated thyroid cancer cell lines expressing PTPN22 protein. The screening results of the present invention are as follows Figure 1 As shown, the IHH4 cell line exhibited expression of PTPN22 protein.

[0056] (2) Screening of monomer compounds in total glycosides of white peony

[0057] The present invention constructs a molecular docking model based on PTPN22, with the aim of screening monomeric compounds in total glucosides of white paeony. Through docking experiments, the present invention found that four compounds in total glucosides of white paeony, including paeoniflorin, paeoniflorin, benzoylpeoniflorin, and hydroxypeoniflorin, all have hydrogen bond interactions with the PTP domain of the receptor PTPN22. Except for paeoniflorin, the other three compounds all show targeted binding to the active residues of PTPN22. The docking score of benzoylpeoniflorin with PTPN22 is better than that of the positive compound L75NO4, while the docking scores of the remaining compounds are similar to that of L75NO4, indicating that benzoylpeoniflorin can be used as a PTPN22 inhibitor. The result of the molecular docking model based on PTPN22 in the present invention is shown in the figure below. Figure 2 As shown, a is the docking result of the active site of PTPN22 and its inhibitor L75NO4; b is the docking result of PTPN22 and paeoniflorin; c is the docking result of PTPN22 and paeoniflorin; d is the docking result of PTPN22 and benzoylpeoniflorin; e is the docking result of PTPN22 and hydroxypeoniflorin. The specific docking results are shown in Table 1.

[0058] Table 1 Specific docking results based on the molecular docking model of PTPN22

[0059]

[0060] (3) Thermal stability test of PTPN22 protein

[0061] The present invention uses a cellular thermal shift assay (CETSA) to evaluate the thermal stability of PTPN22 protein. In this experiment, the present invention constructs an expression plasmid containing the PTPN22 gene and transiently transfects it into the tool cell HEK-293T. 48 hours after transfection, the cells are passaged into two culture dishes for subsequent experiments. After the cells adhere to the wall, they are randomly divided into a control group and an experimental group. The cells in the control group will be treated with dimethyl sulfoxide (DMSO), while the cells in the experimental group will be treated with 300 μM concentration of benzoylpeonidin. After the drug treatment lasts for two hours, the cell samples are collected and each group is divided into 4 equal cell sample groups. The temperature is precisely controlled by a PCR instrument, and the samples of the control group and the experimental group are subjected to a 3-minute thermal cycle treatment at 37°C, 41°C, 44°C, and 47°C, respectively, and each temperature treatment is restored at room temperature for 3 minutes. Subsequently, the sample is placed in liquid nitrogen for rapid freezing, thawed at room temperature, and the freeze-thaw process is repeated three times to achieve cell lysis. Finally, the samples were centrifuged at 12,000 revolutions per minute (rpm) for 10 minutes, and the proteins in the supernatant were collected for Western blot experiments to detect the expression level of the target protein PTPN22. Figure 3 As shown, + represents the treatment with benzoylpeonidin, - represents the treatment without benzoylpeonidin, and benzene represents benzoylpeonidin, and the dosage is 300 μM if used. The experimental results show that after binding with benzoylpeonidin, the proportion of PTPN22 protein that has not been degraded under the same temperature conditions increases, indicating that benzoylpeonidin may interact with PTPN22 protein.

[0062] (4) Binding affinity detection

[0063] The present invention uses surface plasmon resonance (SPR) technology to quantitatively analyze the interaction between PTPN22 protein and benzoylpeonidin. In the experiment, benzoylpeonidin with concentrations of 125μM, 62.5μM, 31.25μM, 15.625μM, and 7.8125μM was introduced into the CM5 chip to monitor the binding process of benzoylpeonidin and PTPN22 protein in real time. The CM5 chip is from cytiva, and the binding affinity test results are as follows Figure 4 As shown, a is the relative corresponding value at different concentrations, and b is the relative corresponding value at different times. Based on steady-state kinetic analysis, the present invention successfully determined the equilibrium dissociation constant (KD value) between the two. The results showed that the binding affinity (KD) of benzoylpeonidin to PTPN22 protein was 42.6 μM.

[0064] (5) Effect of benzoylpeonidin on PTPN22 protein degradation

[0065] Benzoylpaeoniflorin regulates the expression of PTPN22 protein: The IHH4 cell line was isolated at a number of 3×10 6 The cells were plated in a 6 cm dish and divided into an experimental group and a control group after the cells adhered to the wall. The experimental group treated the cell line with 300 μM benzoylpeonidin and incubated for 48 h. Subsequently, the protein samples were collected and the expression level of PTPN22 protein was quantitatively analyzed using Western blot technology. The experimental results are shown in Figure 2. Figure 5 As shown in the figure, benzene represents benzoylpeonidin and β-actin is the internal reference protein. The experimental results show that under the condition of benzoylpeonidin treatment, the expression level of PTPN22 protein is significantly reduced. This phenomenon indicates that benzoylpeonidin may play a role by promoting the degradation of PTPN22 protein.

[0066] Effect of ribosomal inhibitors on benzoylpeonidin-induced PTPN22 protein degradation: The present invention adopts a systematic approach to evaluate the effect of benzoylpeonidin on PTPN22 protein degradation. The systematic method experiment of the present invention involves the addition of 3×10 6 The IHH4 cell line was plated in a 6 cm dish, and after the cells adhered overnight, they were exposed to a 300 μM benzoylpeonidin environment for 48 hours. During this process, the present invention introduced a 100 μM ribosomal inhibitor cycloheximide (CHX) at time points of 0, 6, 12, 18, 21, and 24 hours to inhibit protein synthesis. Through this design, the purpose of the present invention is to explore the specific effects of benzoylpeonidin on the stability of PTPN22 protein when CHX intervenes at different time points. The experimental results are as follows Figure 6 As shown, benzene represents benzoylpaeoniflorin and GAPDH is the internal reference protein.

[0067] PTPN22 protein half-life detection: The present invention uses the proteasome inhibitor MG132 to treat a specific cell line, IHH4. In this experiment, the present invention pre-incubates 4.5×10 6 The IHH4 cell line was plated in a 6 cm dish. After the cells adhered overnight, MG132 at a concentration of 20 μM was added at 0 h, 3 h, 6 h, and 12 h. After 12 h, the protein sample was collected and the expression level of PTPN22 protein was detected by subsequent Western blot experiments. The purpose was to explore the degradation kinetics of PTPN22 protein. By quantitatively analyzing the stability of PTPN22 protein after MG132 treatment, the present invention determined that the optimal enrichment time of PTPN22 protein was 6 hours. The experimental results are as follows Figure 7As shown, β-actin is the internal reference protein.

[0068] Co-IP verifies that benzoylpeonidin ubiquitin degrades PTPN22 protein: The present invention uses co-transfection technology to transfect the plasmid pRK5-HA-Ub containing the HA-Ub tag and the PTPN22 expression plasmid Flag-PTPN22 containing the Flag tag in HEK-293 tool cells. After 8 hours of transfection, the culture medium was replaced to remove the transfection system, and the cells were transferred to two plates after waiting for 40 hours. After the cells adhered to the wall, they were randomly divided into two groups: an experimental group and a control group. The cell lines in the experimental group were continuously exposed to a 300 μM benzoylpeonidin environment after transfection, and the control group was treated with DMSO for 48 hours. In this process, the present invention used 100 μM of the proteasome inhibitor MG132 to synergistically treat the experimental group and the control group cells for 6 hours to block the protein degradation pathway, and then collected protein samples for subsequent analysis. The collected protein samples were quantified and divided into two groups: an input group and an IP group. The samples in the IP group were incubated with Flag magnetic beads at 4°C overnight for subsequent immunoprecipitation experiments. Finally, all samples were processed by Western blot to detect the expression level and ubiquitination status of PTPN22 protein. Figure 8 As shown, IB is immunoblotting, which refers to transferring the protein complex captured by the immunoprecipitation (IP) step to a membrane, and then detecting it with a specific antibody to confirm whether a specific protein is precipitated, + represents addition, - represents no addition, and HA is a tag that can be fused with the protein to express instead of detecting the protein itself. The analysis of the experimental results showed that in cells treated with benzoylpeonidin, the accumulation of HA-Ub tags was significantly higher than that of the control group, indicating that benzoylpeonidin regulates the level of intracellular PTPN22 protein by promoting the ubiquitination of PTPN22 protein, thereby causing its degradation. These results indicate that benzoylpeonidin can be used as a PTPN22 protein degradation regulator.

[0069] (6) Effects of benzoylpeonidin on NIS expression and iodine uptake function in thyroid cancer cells

[0070] Effect of benzoylpeonidin on NIS protein expression

[0071] Total cell protein

[0072] The present invention conducted a dose-response study of benzoylpeonidin on the IHH4 cell line. Specifically, 3×10 6The cells were plated in 6 cm dishes, a total of 6 dishes, and after the cells adhered to the wall, the cell lines were continuously exposed to 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM benzoylpeonidin environments for 48 hours. After the treatment, the present invention extracted total protein from these cells and measured the expression level of NIS protein using Western blot protein quantification technology. The total protein experimental results are shown in Figure 2. Fig. 9 As shown in the figure, benzene represents benzoylpeonidin and β-actin is the internal reference protein. The experimental results revealed a dose-dependent effect: as the concentration of benzoylpeonidin increased, the expression level of NIS protein increased significantly, indicating that benzoylpeonidin has a positive regulatory effect on NIS protein expression.

[0073] Cell membrane proteins

[0074] In order to explore the effect of benzoylpeonidin on the upregulation of NIS protein expression and its localization on the cell membrane, the present invention used a commercial membrane protein extraction kit to separate and extract cell membrane proteins from cells treated with 300 μM benzoylpeonidin. Through protein quantitative analysis techniques such as Western blot, the present invention analyzed the NIS protein in the extracted cell membrane protein samples and the internal reference protein NA of the membrane protein. + / K + The expression levels of NIS protein and the internal reference protein NA of membrane protein were evaluated. + / K + -ATPase expression results are as follows Fig.10 As shown in the figure, benzene represents benzoylpeonidin. The experimental results showed that benzoylpeonidin treatment significantly enhanced the expression of NIS protein on the cell membrane. This indicates that benzoylpeonidin can increase the expression of NIS protein on the cell membrane, thereby enhancing the iodine uptake ability of thyroid cancer cells, which has potential clinical significance for reversing the iodine uptake of iodine-resistant thyroid cancer.

[0075] Effects of benzoylpeonidin on iodine uptake function

[0076] The present invention adopts a chemical analysis method to quantitatively determine the iodine uptake capacity of cells. 6IHH4 cells were plated in 10 cm dishes and divided into two dishes. The experimental group and the control group were treated with benzoylpeonidin and DMSO, respectively. After 48 h, the drug-containing culture medium was removed and the cells were washed three times with Hanks balanced solution containing a final concentration of 10 mM Hepes (collectively referred to as Hanks Buffer), adding 8 mL each time and placed on a shaker at room temperature for 5 min. After washing, Hanks Buffer containing a final concentration of 10 μM NaI was added and incubated at 20°C for 2 h in the dark. After incubation, the cells were washed three more times with pre-cold (4°C) Hanks Buffer, each time on a shaker at room temperature for 5 min. After washing, Hanks Buffer was removed. Buffer, add 500 μL of 100 mM NaOH solution, place on ice to lyse cells, then collect protein samples, centrifuge at 12000 rpm for 15 min, collect supernatant, extract 400 μL of each sample, add 400 μL of sodium arsenite and 400 μL of ammonium cerium sulfate, and incubate at 20°C for 30 min. This involves mixing cells with ammonium cerium sulfate, sodium arsenite, and radioactive iodine isotopes (I - ) and then the absorbance was measured by spectrophotometry at a wavelength of 420 nm to indirectly assess the intracellular iodide content. Fig.11 As shown, ctrl+NaI is the control group, Ben+NaI represents the experimental group using benzoylpeonidin, and the experimental results revealed that benzoylpeonidin significantly enhanced the iodide ion accumulation of thyroid cancer cell lines, indicating that benzoylpeonidin can promote iodine uptake and reverse iodine resistance in thyroid cancer cell lines.

[0077] (7) Exploration of the molecular mechanism of PTPN22 regulating NIS expression

[0078] Molecular Docking Model

[0079] By using computational biology methods, the present invention constructs a molecular docking model of PTPN22 and NIS to predict and simulate the direct interaction between the two. Fig.12 Through docking simulation, the present invention found that there are multiple potential binding sites between the two, and predicted the existence of interactions such as hydrogen bonds and hydrophobic interactions.

[0080] Co-immunoprecipitation (Co-IP) experiments

[0081] The present invention successfully constructed a NIS expression plasmid (Flag-NIS) containing a Flag tag and a PTPN22 expression plasmid (pCDNA3.1-HA-PTPN22) containing an HA tag. The experiment was divided into two groups: an experimental group and a control group. In the experimental group, the two plasmids were co-transfected into the tool cell HEK-293 to achieve co-expression of PTPN22 and NIS; the control group was transfected with an empty vector (pCDNA3.1-HA-empty) of the HA tag and Flag-NIS to exclude the interference of nonspecific interactions. 72 hours after transfection, the present invention collected the whole cell protein supernatant of the cells and performed quantitative analysis to ensure the consistency of the protein amount in subsequent experiments. The collected protein supernatant was divided into an IP group and an input group. In the IP group, the present invention used HA magnetic beads that specifically bind to the HA tag and incubated with the protein supernatant overnight at 4°C to precipitate PTPN22 with the HA tag and its possible interacting proteins. Subsequently, the relevant target protein was detected by Western blot experiment. In the IP group, the presence of NIS in the precipitated protein complex was detected using a specific antibody to verify the direct interaction between PTPN22 and NIS. Fig.13 As shown, IB is immunoblotting, which refers to transferring the protein complex captured by the immunoprecipitation (IP) step to a membrane, and then detecting it with a specific antibody to confirm whether a specific protein is precipitated. + represents addition, - represents no addition, and HA is a tag that can be fused with the protein to replace the detection protein itself. The experimental results showed that HA magnetic beads successfully pulled out the target protein NIS with a Flag tag, indicating that there may be a direct interaction between PTPN22 and NIS.

[0082] Verification of NIS protein expression levels after knockdown / overexpression of PTPN22

[0083] In this experiment, RNA interference (RNAi) technology was used to specifically reduce the expression level of PTPN22 protein in the target cells through small interfering RNA (siRNA). At the same time, by transfecting an expression vector containing the PTPN22 coding sequence, PTPN22 was overexpressed in the target cells, thereby increasing the content of PTPN22 protein. The experimental results show that after the expression of PTPN22 was downregulated, the expression level of NIS protein in the cells was significantly increased. On the contrary, in cells overexpressing PTPN22, the content of NIS protein decreased significantly. This shows that benzoylpeonidin may reverse the content of NIS in iodine-resistant thyroid cancer by downregulating the expression level of PTPN22 and thereby increasing the expression of NIS protein. Specifically, the present invention achieves effective "knockout" of PTPN22 protein expression through siRNA experimental optimization, and analyzes the impact caused by the reduction in target protein concentration. The experimental results are as follows Fig.14 As shown, siNC is the control siRNA of the siPTPN22 group, ctrl is the control plasmid of the Over-PTPN22 group, siPTPN22 is the siRNA that knocks down the target gene PTPN22, and over-PTPN22 is the plasmid that overexpresses the target gene PTPN22. The experimental results revealed a negative correlation between PTPN22 and NIS expression, indicating that benzoylpeonidin can be used in drugs to reverse iodine resistance in thyroid cancer.

[0084] Example 1: A Chinese medicine monomer

[0085] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, the adjuvant is a medical solvent, and the medical solvent is DMSO. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent.

[0086] Example 2: A Chinese medicine monomer

[0087] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, the adjuvant is a medical solvent, and the medical solvent is ethanol. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent.

[0088] Example 3: A Chinese medicine monomer

[0089] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, and the adjuvant is a medical solvent, wherein the medical solvent is ethanol and polyoxyethylene castor oil, wherein the ethanol and polyoxyethylene castor oil are mixed in a mass ratio of 1:0.1. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent.

[0090] Example 4: A Chinese medicine monomer

[0091] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, the adjuvant is a medical solvent and an adjuvant, and the medical solvent is DMSO. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent. The adjuvant is an amino alcohol benzoate compound, and the amount of the amino alcohol benzoate compound used is 0.008wt% of the medical solvent.

[0092] Preparation of amino alcohol benzoate compound: 3-hydroxybenzoic acid ethyl ester, anhydrous potassium carbonate and potassium iodide are added to DMF and stirred and mixed, epichlorohydrin is added at 30°C, pH is adjusted to 10, and then the reaction is carried out at 150°C for 6 hours. After the reaction is completed, the intermediate product is separated by column chromatography, the intermediate product is mixed with ethanol, an amino alcohol compound is added, and the reaction is carried out at 80°C for 6 hours. Water is added to precipitate to obtain an amino alcohol benzoate compound. The amount of 3-hydroxybenzoic acid ethyl ester used is 10wt% of DMF, the amount of anhydrous potassium carbonate used is 40wt% of 3-hydroxybenzoic acid ethyl ester, the amount of potassium iodide used is 30wt% of 3-hydroxybenzoic acid ethyl ester, the amount of epichlorohydrin used is 50wt% of 3-hydroxybenzoic acid ethyl ester, the amount of the intermediate product used is measured based on 3-hydroxybenzoic acid ethyl ester, the amino alcohol compound is ethyl 2-aminoacetate, and the amount of the amino alcohol compound used is 80wt% of 3-hydroxybenzoic acid ethyl ester.

[0093] Example 5: A Chinese medicine monomer

[0094] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, the adjuvant is a medical solvent and an adjuvant, and the medical solvent is ethanol. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent. The adjuvant is an amino alcohol benzoate compound, and the amount of the amino alcohol benzoate compound used is 0.008wt% of the medical solvent.

[0095] Example 6: A Chinese medicine monomer

[0096] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, and the adjuvant is a medical solvent and an adjuvant. The medical solvent is ethanol and polyoxyethylene castor oil, and the ethanol and polyoxyethylene castor oil in the medical solvent are mixed at a mass ratio of 1:0.1. The amount of benzoylpeonidin used is 0.008wt% of the medical solvent. The adjuvant is an amino alcohol benzoate compound, and the amount of the amino alcohol benzoate compound used is 0.008wt% of the medical solvent.

[0097] Example 7: A Chinese medicine monomer

[0098] The difference between this embodiment and embodiment 1 is that the amount of benzoylpeonidin used is 0.015 wt % of the medical solvent.

[0099] Example 8: A Chinese medicine monomer

[0100] The difference between this embodiment and embodiment 2 is that the amount of benzoylpeonidin used is 0.015 wt % of the medical solvent.

[0101] Example 9: A Chinese medicine monomer

[0102] The difference between this embodiment and embodiment 3 is that the amount of benzoylpeonidin used is 0.015 wt % of the medical solvent.

[0103] Example 10: A Chinese medicine monomer

[0104] The difference between this embodiment and embodiment 4 is that the usage amount of benzoylpeonidin is 0.015wt% of the medical solvent.

[0105] Example 11: A Chinese medicine monomer

[0106] The difference between this embodiment and embodiment 5 is that the amount of benzoylpeonidin used is 0.015 wt % of the medical solvent.

[0107] Example 12: A Chinese medicine monomer

[0108] The difference between this embodiment and embodiment 6 is that the amount of benzoylpeonidin used is 0.015 wt % of the medical solvent.

[0109] Example 13: A Chinese medicine monomer

[0110] The difference between this embodiment and embodiment 1 is that the amount of benzoylpeonidin used is 0.021 wt % of the medical solvent.

[0111] Example 14: A Chinese medicine monomer

[0112] The difference between this embodiment and embodiment 2 is that the amount of benzoylpeonidin used is 0.021 wt % of the medical solvent.

[0113] Example 15: A Chinese medicine monomer

[0114] The difference between this embodiment and embodiment 3 is that the amount of benzoylpeonidin used is 0.021 wt % of the medical solvent.

[0115] Example 16: A Chinese medicine monomer

[0116] The difference between this embodiment and embodiment 4 is that the amount of benzoylpeonidin used is 0.021 wt % of the medical solvent.

[0117] Example 17: A Chinese medicine monomer

[0118] The difference between this embodiment and embodiment 5 is that the amount of benzoylpeonidin used is 0.021 wt % of the medical solvent.

[0119] Example 18: A Chinese medicine monomer

[0120] The difference between this embodiment and embodiment 6 is that the usage amount of benzoylpeonidin is 0.021wt% of the medical solvent.

[0121] Example 19: A Chinese medicine monomer

[0122] The difference between this embodiment and embodiment 18 is that the auxiliary agent further includes 1-O-p-coumaryl glycerol. The usage amount of 1-O-p-coumaryl glycerol is 0.0006wt% of the medical solvent.

[0123] Example 20: A Chinese medicine monomer

[0124] The difference between this embodiment and embodiment 19 is that the auxiliary agent further includes 1-O-p-coumaryl glycerol. The usage amount of 1-O-p-coumaryl glycerol is 0.001wt% of the medical solvent.

[0125] Comparative Example 1: A Chinese medicine monomer

[0126] A Chinese medicine monomer agent: comprising a Chinese medicine monomer and an adjuvant. The Chinese medicine monomer is benzoylpeonidin, and the adjuvant is a medical solvent and an adjuvant. The medical solvent is ethanol and polyoxyethylene castor oil, and the ethanol and polyoxyethylene castor oil in the medical solvent are mixed at a mass ratio of 1:0.1. The amount of benzoylpeonidin used is 0.0005wt% of the medical solvent. The adjuvant is an amino alcohol benzoate compound, and the amount of the amino alcohol benzoate compound used is 0.008wt% of the medical solvent.

[0127] Comparative Example 2: A Chinese medicine monomer

[0128] Compared with Comparative Example 1, the difference between this comparative example and Comparative Example 1 is that the amount of the Chinese medicine monomer used is 0. The other components and amounts are the same as those in Comparative Example 1.

[0129] Test example:

[0130] The present invention uses cells to test the cytotoxicity of the amino alcohol benzoate compound prepared in Example 4, adds the amino alcohol benzoate compound to DMSO and mixes, the amino alcohol benzoate compound is used in an amount of 300% of DMSO, and is diluted with DMEM culture medium to a content of 0.008wt% of the amino alcohol benzoate compound, and FTC-238 cells are cultured as an experimental group, and a control group is set, in which the amino alcohol benzoate compound is not added, and the other is consistent with the experimental group, and the FTC-238 cells are tested by the CCK-8 method, and the cell proliferation rate of the experimental group and the control group is basically the same, indicating that the amino alcohol benzoate compound is not toxic to the cells. When the amount of the amino alcohol benzoate compound is within the range of 0.0005-0.03wt%, it is also non-toxic to the cells.

[0131] The present invention tests the effect of the Chinese medicine monomer obtained in Examples 6, 12, 18-20 and Comparative Examples 1-2 on cell culture. In this test, 90wt% of the medical solvent in the Chinese medicine monomer obtained in Examples 6, 12, 18-20 and Comparative Examples 1-2 was replaced with DMEM culture medium, and then the IHH4 cell line was cultured for 48 hours, and then a commercial membrane protein extraction kit was used to separate and extract the cell membrane protein. The expression level of NIS protein in the extracted cell membrane protein sample was characterized by protein quantitative analysis techniques such as Western blot. Taking the expression of NIS protein in the experimental group of Comparative Example 2 as 1, the relative expression amount of NIS protein is as follows: Fig.15 As shown, S6 is Example 6, S12 is Example 12, S18 is Example 18, S19 is Example 19, S20 is Example 20, D1 is Comparative Example 1, and D2 is Comparative Example 2. The present invention studies the Chinese medicine monomers including paeoniflorin, paeoniflorin lactone glycosides, benzoylpeoniflorin, and hydroxypeoniflorin. The effect of paeoniflorin lactone glycosides is not good, while paeoniflorin, benzoylpeoniflorin, and hydroxypeoniflorin all have good effects. Taking benzoylpeoniflorin as an example, the use of benzoylpeoniflorin When the amount is 0.001-0.05wt%, the expression of NIS protein can be improved. The present invention also prepares an amino alcohol benzoate compound, which is composed of ethyl 3-hydroxybenzoate, epichlorohydrin and ethyl 2-aminoacetate. When the amino alcohol benzoate compound is compounded with benzoylpeonidin, the expression of NIS protein can be improved. The present invention also finds that if the amount of benzoylpeonidin is too low, the expression of NIS protein cannot be improved. When using the amino alcohol benzoate compound and benzoylpeonidin, 1-O-p-coumarylglycerol can be added to further improve the expression of NIS protein.

[0132] When the dosage of the paeoniflorin and hydroxypeoniflorin disclosed in the present invention is between 0.001-0.05wt%, they also have the effect of increasing the expression of NIS protein.

[0133] The present invention tests the effect of the Chinese medicine monomer obtained in Examples 6, 12, 18-20 and Comparative Examples 1-2 on the iodine uptake ability of cell culture. In this test, 90wt% of the medical solvent in the Chinese medicine monomer obtained in Examples 6, 12, 18-20 and Comparative Examples 1-2 is replaced with DMEM culture medium, and then the IHH4 cell line is cultured for 48h, and then the iodine uptake ability is tested according to the chemical analysis method in the research content. The results of iodine ion content are as follows: Fig.16As shown, S6 is Example 6, S12 is Example 12, S18 is Example 18, S19 is Example 19, S20 is Example 20, D1 is Comparative Example 1, and D2 is Comparative Example 2. The present invention studies the Chinese medicine monomers including paeoniflorin, paeoniflorin lactone glycosides, benzoylpeoniflorin, and hydroxypeoniflorin. The effect of paeoniflorin lactone glycosides is not good, while paeoniflorin, benzoylpeoniflorin, and hydroxypeoniflorin all have good effects. Taking benzoylpeoniflorin as an example, in benzoylpeoniflorin When the dosage is 0.001-0.05wt%, the iodine uptake capacity of the cell can be improved. The present invention also prepares an amino alcohol benzoate compound, which is composed of ethyl 3-hydroxybenzoate, epichlorohydrin and ethyl 2-aminoacetate. When the amino alcohol benzoate compound is compounded with benzoylpeonidin, the iodine uptake capacity of the cell can be improved. The present invention also finds that if the dosage of benzoylpeonidin is too low, the iodine uptake capacity of the cell cannot be improved. When using the amino alcohol benzoate compound and benzoylpeonidin, 1-O-p-coumarylglycerol can also be added to further improve the iodine uptake capacity of the cell.

[0134] When the dosage of the paeoniflorin and hydroxypeoniflorin disclosed in the present invention is between 0.001-0.05wt%, the iodine uptake capacity of cells can also be improved.

[0135] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0136] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A Chinese medicine monomer, comprising: Traditional Chinese medicine monomer and adjuvant; the traditional Chinese medicine monomer includes benzoylpeonidin, the adjuvant includes at least a medical solvent, and the medical solvent includes at least one of DMSO, ethanol, isopropanol, acetone, benzyl alcohol, ethyl acetate, polyoxyethylene castor oil and water; the amount of the traditional Chinese medicine monomer used is 0.001-0.05wt% of the medical solvent; The auxiliary agent also includes an auxiliary agent, which includes an amino alcohol benzoate compound, and the amino alcohol benzoate compound is formed by combining ethyl 3-hydroxybenzoate, epichlorohydrin and ethyl 2-aminoacetate; In the preparation of the amino alcohol benzoate compound, ethyl 3-hydroxybenzoate, anhydrous potassium carbonate and potassium iodide are added to DMF and stirred and mixed, epichlorohydrin is added at 20-40° C., the pH is adjusted to 9-10, and then the reaction is carried out at 120-180° C. for 3-12 hours. After the reaction is completed, the intermediate product is separated by column chromatography, the intermediate product is mixed with ethanol, the amino alcohol compound is added, the reaction is carried out at 70-90° C. for 3-12 hours, and water is added to precipitate to obtain the amino alcohol benzoate compound; the amino alcohol compound is ethyl 2-aminoacetate; The auxiliary agent also includes 1-O-p-coumaryl glycerol, and the usage amount of 1-O-p-coumaryl glycerol is 0.0001-0.003wt% of the medical solvent.

2. A Chinese medicine monomer according to claim 1, characterized in that: The medical solvent is DMSO; or, the medical solvent is ethanol; or, the medical solvent is ethanol and polyoxyethylated castor oil; or, the medical solvent is isopropanol; or, the medical solvent is ethyl acetate.

3. A Chinese medicine monomer according to claim 2, characterized in that: The ethanol and polyoxyethylated castor oil in the medical solvent are mixed in a mass ratio of 1:0.01-0.

3.

4. Use of a Chinese medicine monomer according to any one of claims 1 to 3 in the preparation of a drug for treating thyroid cancer.

5. Use of a Chinese medicine monomer according to any one of claims 1 to 3 in the preparation of a drug for treating iodine-resistant thyroid cancer.

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