Use of NDV in preparation of medicine for treating hyperthyroidism

Abstract

This invention provides the application of NDV in the preparation of drugs for treating hyperthyroidism, belonging to the field of pharmaceutical technology. This invention discovers that the cytotoxic effect of NDV is significantly time-dependent, with the killing effect gradually increasing with prolonged culture time. Furthermore, this invention is the first to discover that NDV can significantly enhance the cytotoxic ability of JAK inhibitors against thyroid follicular epithelial cells, effectively inhibit the growth of overactive thyroid cells, and induce apoptosis. This not only overcomes the limitation of JAK inhibitors alone in killing thyroid epithelial cells, but also reduces the cells' natural resistance to NDV by inhibiting the JAK-STAT pathway, thus strengthening the lytic effect of oncolytic viruses on hyperactive thyroid cells, providing a novel drug combination strategy with a synergistic mechanism for the treatment of hyperthyroidism.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of NDV in the preparation of drugs for treating hyperthyroidism. Background Technology

[0002] Hyperthyroidism is a common endocrine disorder characterized by excessive synthesis and secretion of thyroid hormones, leading to hypermetabolism and sympathetic nerve excitation. The etiology of hyperthyroidism is complex, with Graves' disease being the most common. Graves' disease is essentially an autoimmune disorder that triggers excessive stimulation of thyroid follicular epithelial cells, ultimately resulting in abnormally high thyroid hormone secretion. Currently, clinical treatment strategies for hyperthyroidism mainly include antithyroid drug therapy, radioactive iodine-131 therapy, and surgical resection. However, all of these existing treatments have significant limitations: antithyroid drug therapy has a long treatment cycle, a high relapse rate, and may cause adverse reactions such as liver damage and agranulocytosis; radioactive iodine-131 therapy easily leads to permanent hypothyroidism; and surgery carries risks such as trauma, recurrent laryngeal nerve damage, and hypoparathyroidism. Therefore, exploring novel, minimally invasive, and selective methods for removing overactive thyroid cells is of significant clinical importance.

[0003] Newcastle disease virus (NDV) is a negative-sense RNA virus with oncolytic activity that selectively infects and kills various tumor cells while exhibiting low cytotoxicity to normal cells. Tumor cells often exhibit defects in antiviral signaling pathways, such as abnormal interferon (IFN) responses or JAK-STAT signaling inhibition, allowing NDV to replicate efficiently and induce cell death. NDV is typically used in combination with other drugs to enhance the therapeutic effect in cancer treatment; however, there are currently no clear reports of oncolytic viruses being used to treat hyperthyroidism.

[0004] Based on the current state of the technology, existing treatments for hyperthyroidism all have insurmountable limitations, failing to achieve the therapeutic goal of novel, minimally invasive, and selective elimination of overactive thyroid cells. Therefore, how to overcome existing technological bottlenecks, improve the efficiency of killing hyperactive thyroid cells, and develop a novel treatment strategy for hyperthyroidism has become a key technical problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of NDV in the preparation of medicaments for treating hyperthyroidism.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of NDV in the preparation of drugs for treating hyperthyroidism.

[0007] This invention provides the application of NDV in the preparation of drugs that enhance the anti-hyperthyroidism efficacy of JAK inhibitors.

[0008] Preferably, the NDV is a non-virulent or attenuated Newcastle disease virus strain.

[0009] Preferably, the JAK inhibitor includes AG490 or ruxolitinib.

[0010] This invention provides a drug for treating hyperthyroidism, the drug comprising NDV; The final concentration of NDV in the drug is 1~100 MOI.

[0011] Preferably, the drug further includes a JAK inhibitor; The JAK inhibitors include AG490 or ruxolitinib.

[0012] Preferably, the final concentration of AG490 in the drug is 1~100 nM.

[0013] Preferably, the final concentration of ruxolitinib in the drug is 0.37~9.9 nM.

[0014] Preferably, the dosage form of the drug includes an injection.

[0015] Preferably, the drug includes pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of NDV in the preparation of drugs for treating hyperthyroidism, revealing that the cytotoxic effect of NDV is significantly time-dependent, with the killing effect gradually increasing with prolonged culture time. This invention also reveals for the first time, through in vitro and in vivo experiments, the synergistic effect of NDV and JAK inhibitors in the treatment of hyperthyroidism. Experimental results show that NDV can significantly enhance the cytotoxic ability of JAK inhibitors against thyroid follicular epithelial cells, effectively inhibit the growth of overactive thyroid cells, and induce apoptosis. This combined treatment regimen not only overcomes the limitation of JAK inhibitors alone in killing thyroid epithelial cells, but also reduces the cells' natural resistance to NDV by inhibiting the JAK-STAT pathway, thus strengthening the lytic effect of oncolytic viruses on hyperactive thyroid cells, providing a novel drug combination strategy with a synergistic mechanism for the treatment of hyperthyroidism. Attached Figure Description

[0017] Figure 1 Microscopic morphological observations of Nthy-ori 3-1 cells after treatment with NDV at different MOI gradients; Figure 2Microscopic morphological observations of Nthy-ori 3-1 cells after NDV treatment at different infection times; Figure 3 The figure shows the results of CCK-8 assay to detect the effect of different MOI NDV on the viability of Nthy-ori 3-1 cells; Figure 4 The figure shows the results of CCK-8 assay to detect the effect of NDV on the viability of Nthy-ori 3-1 cells at different treatment times; Figure 5 Scatter plot for flow cytometry detection of Nthy-ori 3-1 cell apoptosis induced by different MOI NDV; Figure 6 A statistical analysis of the apoptosis rate of Nthy-ori 3-1 cells under different MOI NDV treatments; Figure 7 Fluorescence micrographs of EdU staining to detect the effect of different MOI NDV on the proliferation of Nthy-ori 3-1 cells; Figure 8 A statistical chart showing the proportion of EdU-positive cells in Nthy-ori 3-1 cells after different MOI NDV treatments; Figure 9 Volcano plot of differentially expressed genes after NDV infection of Nthy-ori 3-1 cells; Figure 10 GO functional enrichment analysis of differentially expressed genes after NDV infection of Nthy-ori 3-1 cells; Figure 11 A diagram showing the KEGG pathway enrichment analysis of differentially expressed genes after NDV infection of Nthy-ori 3-1 cells; Figure 12 A heatmap showing the expression of differentially expressed genes in the JAK-STAT pathway after NDV infection of Nthy-ori 3-1 cells; Figure 13 The figure shows the results of qPCR validation of the expression levels of key genes in the JAK-STAT pathway after NDV infection; Figure 14 Figure 1 shows the viability of Nthy-ori 3-1 cells after treatment with different concentrations of AG490 and Ruxolitinib. Figure 15 The image shows the viability of Nthy-ori 3-1 cells after treatment with NDV combined with a JAK inhibitor. Figure 16 Flow cytometry scatter plot of apoptosis in Nthy-ori 3-1 cells after treatment with NDV combined with JAK inhibitor; Figure 17A graph showing the statistical results of apoptosis rate in Nthy-ori 3-1 cells after treatment with NDV combined with a JAK inhibitor; Figure 18 EdU fluorescence microscopy image of Nthy-ori 3-1 cells after treatment with NDV combined with JAK inhibitor; Figure 19 A statistical result of the proportion of EdU-positive cells after Nthy-ori 3-1 cells were treated with NDV combined with a JAK inhibitor; Figure 20 The ELISA standard curve for detecting serum thyroxine (T4) in mice and the results of T4 level detection in each group are shown in the figure. Figure 21 The ELISA standard curve for detecting serum thyroid-stimulating hormone (TSH) in mice and the results of TSH level detection in each group are shown in the figure. Figure 22 Images showing the HE staining results of thyroid tissue from each group of mice; Figure 23 The image shows the results of Ki67 immunohistochemical staining of thyroid tissue from mice in each group. Detailed Implementation

[0018] This invention provides the application of NDV in the preparation of drugs for treating hyperthyroidism.

[0019] In this invention, the NDV is preferably a non-toxic or attenuated Newcastle disease virus strain.

[0020] This invention provides the application of NDV in the preparation of drugs that enhance the anti-hyperthyroidism efficacy of JAK inhibitors.

[0021] In this invention, the NDV is preferably a non-toxic or attenuated Newcastle disease virus strain; the JAK inhibitor preferably includes AG490 or ruxolitinib.

[0022] This invention provides a drug for treating hyperthyroidism, the drug comprising NDV.

[0023] In this invention, the final concentration of the NDV in the drug is preferably 1-100 nM, more preferably 3-50 nM, and even more preferably 5 nM; the drug preferably also includes a JAK inhibitor; the JAK inhibitor preferably includes AG490 or ruxotinib. The final concentration of AG490 in the drug is preferably 1-100 nM, more preferably 5-50 nM, and even more preferably 10 nM; the final concentration of ruxotinib in the drug is preferably 0.37-9.9 nM, more preferably 1.1-5.5 nM, and even more preferably 3 nM.

[0024] In this invention, the dosage form of the drug preferably includes an injection; the drug preferably includes pharmaceutically acceptable excipients.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Source of materials

[0027] NDV virus, amplified and cultured in chicken embryos, and preserved in the laboratory; Human thyroid normal cell line Nthy-ori 3-1, preserved in the laboratory; Serum-free culture medium was purchased from Abcell; The complete culture medium was purchased from Abcell; CCK-8 test solution was purchased from Seven Biotechnology Co., Ltd. PI staining solution was purchased from Shanghai Yisheng Biotechnology Co., Ltd. The binding buffer was purchased from Shanghai Yisheng Biotechnology Co., Ltd. EdU cell proliferation assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. TRIzol reagent was purchased from Baori Biotechnology (Beijing) Co., Ltd. Chloroform was purchased from Sinopharm Chemical Reagent Co., Ltd. Isopropanol was purchased from Tianjin Jindong Tianzheng Fine Chemical Reagent Factory. The reverse transcription kit was purchased from Takara Bio Inc., Dalian. The JAK inhibitors AG490 and ruxolitinib were purchased from TargetMOI. SPF-grade female C57BL / 6 mice were purchased from Henan Skbes Biotechnology Co., Ltd. The T4 and TSH ELISA kits were purchased from Wuhan Saipei Biotechnology Co., Ltd. HE staining and Cleaved Caspase-3 (CC3) immunohistochemical staining agents were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0028] Example 1

[0029] (111) Experimental Groups: (1.1.1) Dose-dependent experimental grouping: Control group (CON group): Normally cultured Nthy-ori 3-1 cells; NDV-L group: Nthy-ori 3-1 cells were infected with NDV at MOI=1; NDV-M group: Nthy-ori 3-1 cells were infected with NDV at MOI=5; NDV-H group: Nthy-ori 3-1 cells were infected with NDV at MOI=10; (1.1.2) Time-dependent experimental grouping: 0h group: Nthy-ori 3-1 cells infected with NDV at MOI=1 0h post-infection; 24h group: Nthy-ori 3-1 cells infected with NDV at MOI=1 24h; 48h group: Nthy-ori 3-1 cells infected with NDV at MOI=1 48h; 72h group: Nthy-ori 3-1 cells infected with NDV at MOI=1 72h.

[0030] (1.2) Effects of NDV on the morphology, viability, apoptosis and proliferation of Nthy-ori 3-1 cells

[0031] (1.2.1) Cell seeding

[0032] Nthy-ori 3-1 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 100 μL of cells per well; seeded in 6-well plates at a final volume of 100 μL per well. 5 10 cells per well, with a final volume of 2 mL per well; incubate at 37℃ and 5% CO2 for 12-24 h until the cells are fully adhered.

[0033] (1.2.2) Virus handling

[0034] Dose-dependent group: The original culture medium was discarded and serum-free NDV culture medium containing MOI=1, 5 and 10 was added respectively. After incubation for 2 hours, the medium was replaced with complete culture medium and cultured for 24 hours. The culture was then photographed under a microscope. Time-dependent group: Cells were infected with NDV with MOI=1, and cells were collected at 0h, 24h, 48h and 72h after infection for testing and microscopic imaging.

[0035] (1.2.3) CCK-8 assay for cell viability

[0036] Add 10 μL of CCK-8 detection solution to each well, gently shake to mix, and incubate in a 37℃, 5% CO2 incubator in the dark for 2 h; use an ELISA reader to measure the absorbance (OD) value of each well at a wavelength of 450 nm.

[0037] (1.2.4) Flow cytometry detection of apoptosis

[0038] Collect cell supernatants and adherent cells from each group, combine them into centrifuge tubes, wash twice with pre-cooled PBS, and centrifuge at 1000 r / min for 5 min; discard the supernatant, add 100 μL of binding buffer to resuspend the cells; add 10 μL of PI staining solution, mix gently, and incubate at room temperature in the dark for 15 min; after incubation, add 400 μL of binding buffer, and immediately use flow cytometry to detect the apoptosis rate.

[0039] (1.2.5) EdU detection of cell proliferation

[0040] Add EdU working solution to each group of cells and incubate in an incubator for 1 hour; discard the culture medium, add fixative and fix at room temperature for 15 minutes; discard the fixative, add permeabilization solution and incubate at room temperature for 15 minutes; add Click reaction solution and incubate in the dark for 30 minutes; counterstain cell nuclei with DAPI in the dark for 10 minutes; observe and photograph using a fluorescence microscope, and count the proportion of EdU positive cells.

[0041] (1.3) Experimental Results

[0042] Experimental results are as follows Figures 1-8 As shown, NDV can cause Nthy-ori 3-1 cells to shrink, detach, and die, and inhibit cell viability, induce apoptosis, and inhibit cell proliferation in a dose-dependent manner. At the same time, under the condition of fixed MOI=1, the killing effect of NDV on cells is significantly time-dependent, and the killing effect gradually increases with the extension of culture time, but the overall killing effect of NDV alone is limited.

[0043] Example 2

[0044] (2.1) Experimental Grouping

[0045] Control group: Nthy-ori 3-1 cells cultured normally; NDV group: Nthy-ori 3-1 cells were infected with NDV at MOI=1.

[0046] (2.2) Study on the mechanism of NDV killing thyroid cells

[0047] (2.2.1) Cell seeding and treatment

[0048] Nthy-ori 3-1 cells were seeded in 6-well plates at a density of 5 × 10⁶ cells per well. 5 100 cells were added to each well with 2 mL of complete culture medium and incubated at 37°C in a 5% CO2 incubator until the cells adhered. The NDV treatment group was given NDV serum-free medium with an MOI of 1. After 2 h of infection, the medium was replaced with normal complete medium and cultured for another 36 h.

[0049] (2.2.2) Total RNA extraction using the TRIzol method

[0050] Discard the culture medium, add 1 mL of TRIzol reagent to each well, repeatedly pipette to fully lyse the cells, and incubate at room temperature for 5 min; add 200 μL of chloroform to each tube, shake vigorously for 15 s, and incubate at room temperature for 2 min; centrifuge at 12000 rpm for 15 min at 4 °C, and transfer the upper aqueous phase to a new centrifuge tube; add an equal volume of isopropanol, gently invert to mix, and incubate at room temperature for 10 min; centrifuge at 12000 rpm for 10 min at 4 °C, and discard the supernatant; wash the precipitate with 1 mL of 75% ethanol, centrifuge at 7500 rpm for 5 min at 4 °C, and discard the supernatant; air dry the RNA precipitate at room temperature, dissolve it in enzyme-free water, and use a nucleic acid quantification instrument to detect the concentration and purity.

[0051] (2.2.3) Transcriptome sequencing and analysis

[0052] Two groups of three biological replicates of cells were collected, treated for 36 h, and then the cells were collected and total RNA was extracted. After passing quality inspection, the cells were sent to Novogene for transcriptome sequencing and subsequent data analysis, including differentially expressed gene screening, KEGG pathway and GO functional enrichment analysis, with a focus on changes in the JAK-STAT signaling pathway.

[0053] (2.2.4) cDNA synthesis

[0054] Take 1 μg of total RNA, prepare the reaction system according to the reverse transcription kit instructions, mix gently, and place in a PCR instrument for reverse transcription reaction. The reaction conditions are 37℃ for 15 min and 85℃ for 5 s. After the reaction is complete, cDNA is obtained.

[0055] (2.2.5) qPCR detection of gene expression

[0056] Using cDNA as a template and GAPDH as an internal reference gene, a qPCR reaction system was prepared. The amplification program was: 95℃ pre-denaturation for 30s; 95℃ for 5s, 60℃ for 30s, for a total of 40 cycles. The relative expression levels of key genes in the JAK-STAT pathway were calculated using the 2-ΔΔCt method.

[0057] (2.3) Experimental Results

[0058] Experimental results are as follows Figures 9-13 As shown, NDV infection can significantly activate the JAK-STAT signaling pathway in Nthy-ori 3-1 cells, and the expression of related genes is significantly upregulated, indicating that the JAK-STAT pathway is a key mechanism for thyroid cells to resist NDV killing and for NDV monotherapy to be ineffective.

[0059] Example 3

[0060] (3.1) Experimental Grouping

[0061] Control group: Normally cultured Nthy-ori 3-1 cells; AG490 treatment groups: Cells were treated with AG490 at concentrations of 1 nM, 3 nM, 5 nM, 10 nM, and 100 nM, respectively; Ruxolitinib treatment groups: Cells were treated with ruxolitinib at concentrations of 0.37 nM, 1.1 nM, 3.3 nM, and 9.9 nM, respectively.

[0062] (3.2) Effect of JAK inhibitor monotherapy on Nthy-ori 3-1 cell viability

[0063] Nthy-ori 3-1 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured at 37°C in a 5% CO2 incubator for 12-24 hours until fully adherent. The original culture medium was discarded, and complete culture medium containing different concentrations of AG490 or ruxolitinib was added to each well, followed by 24 hours of further culture. After culturing, 10 μL of LCK-8 assay solution was added to each well, gently mixed, and incubated in the dark for 2 hours. The absorbance was then measured at 450 nm using a microplate reader.

[0064] (3.3) Experimental Results

[0065] Experimental results are as follows Figure 14 As shown, AG490 at concentrations of 1-100 nM and ruxolitinib at concentrations of 0.37-9.9 nM had no significant effect on the viability of Nthy-ori 3-1 cells and can be used as safe concentrations for subsequent combined experiments.

[0066] Example 4

[0067] (4.1) Experimental Grouping

[0068] Control group (referred to as CON or Control group): Normally cultured Nthy-ori 3-1 cells; NDV group: Nthy-ori 3-1 cells were infected with NDV at MOI=1; AG490 group: Nthy-ori 3-1 cells were treated with 10 nM AG490; Ruxolitinib group: Nthy-ori 3-1 cells were treated with 3.3 nM Ruxolitinib; Group A+N: Nthy-ori 3-1 cells were treated with NDV at MOI=1 combined with 10 nM AG490; R+N group: Nthy-ori 3-1 cells were treated with NDV with MOI=1 combined with 3 nM Ruxolitinib.

[0069] (4.2) Synergistic killing effect of NDV combined with JAK inhibitor on Nthy-ori 3-1 cells

[0070] (4.2.1) Cell seeding

[0071] Nthy-ori 3-1 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at 5 × 10⁶ cells per well in a 6-well plate. 5 Cells were cultured in a 37°C, 5% CO2 incubator until they adhered to the culture vessel.

[0072] (4.2.2) Cell treatment

[0073] After adding the drugs according to the groups, continue culturing for 24 hours.

[0074] (4.2.3) CCK-8 assay for cell viability

[0075] Add 10 μL of CCK-8 detection solution to each well, mix well, and incubate in the dark for 2 hours. Measure the absorbance at 450 nm using a microplate reader and calculate cell viability.

[0076] (4.2.4) Flow cytometry detection of apoptosis

[0077] Cells were collected, washed twice with PBS, stained with PI, and incubated at room temperature in the dark for 15 min. The apoptosis rate was then detected by flow cytometry.

[0078] (4.2.5) EdU detection of cell proliferation

[0079] After incubation with EdU working solution for 2 hours, followed by fixation, permeabilization, Click reaction staining, and DAPI counterstaining, the proportion of positive cells was observed and counted under a fluorescence microscope.

[0080] (4.3) Experimental Results

[0081] Experimental results are as follows Figures 15-19 As shown, compared with the NDV monotherapy group, NDV combined with JAK inhibitors can significantly reduce Nthy-ori 3-1 cell viability, increase cell apoptosis rate, and inhibit cell proliferation, demonstrating a significant synergistic killing effect.

[0082] Example 5

[0083] (5.1) Experimental Grouping

[0084] Ctrl group: Normal mice were treated with only an equal volume of saline. NDV group: Mice treated with Newcastle disease virus (NDV) only; Ruxo group: Mice treated with Ruxolitinib only; Ruxo+NDV group: Mice were treated with both Newcastle disease virus (NDV) and ruxotinib.

[0085] (5.2) Effects of NDV combined with JAK inhibitor on thyroid function in normal C57BL / 6 mice in vivo

[0086] (5.2.1) Animal adaptation: SPF-grade female C57BL / 6 mice were adapted for 1 week, with free access to food and water, and were kept in an SPF-grade environment with constant temperature and humidity and a balanced diurnal rhythm.

[0087] (5.2.2) Orthotopic injection of thyroid gland: Mice were anesthetized intraperitoneally, and after hair removal and disinfection of the neck, the skin was incised along the midline of the neck, and the subcutaneous tissue and muscle were separated to expose the thyroid tissue; orthotopic injection of thyroid gland was performed according to the grouping protocol: the control group was injected with physiological saline (20 μL / side), and the NDV group was injected with NDV suspension (concentration of 1×10⁻⁶). 7 PFU / mL, 2×10 5 The ruxotinib group received ruxotinib solution (concentration 1 mg / mL, 20 μg / side, embedded in ZIF-8 material, solvent: sterile saline), while the combination group received a mixture of NDV and ruxotinib (NDV concentration 1×10⁻⁶). 7 (PFU / mL, ruxotinib concentration 1 mg / mL, dosage same as the single-drug group); after injection, the muscle layer and skin layer were sutured in layers and disinfected with povidone-iodine. The patient was fed routinely for 2 weeks after the operation.

[0088] (5.2.3) Sample collection: After the intervention period, peripheral blood was collected by capillary needle blood collection, and serum was separated by centrifugation after standing at room temperature. Then, the mice were euthanized by cervical dislocation, the thyroid tissue was quickly dissected, the surface blood was removed by rinsing with pre-cooled sterile PBS, and the tissue was fixed in 4% paraformaldehyde fixative at room temperature.

[0089] (5.2.4) Serum hormone level detection: Take out the frozen serum samples and thaw them naturally on ice. At the same time, take out the mouse T4 and TSH ELISA kits and related reagents and equilibrate at room temperature for 30 min. Follow the instructions in the kit: ① Add samples: Add 50 μL of standard, blank control and serum samples of each group to the corresponding wells of the ELISA plate. Set up 3 replicates for each sample. Add an equal volume of sterile PBS to the blank control wells. ② Seal and incubate: After adding samples, immediately seal the ELISA plate with the sealing film and incubate at 37℃ for 60 min. ③ Wash the plate: After incubation, carefully remove the sealing film, discard the liquid in the wells, and wash each well thoroughly with washing buffer. Discard the residual liquid in the wells after each wash. Repeat the washing 5 times and finally pat dry on absorbent paper. ④ Add enzyme-labeled reagent: Add 50 μL of enzyme-labeled conjugate to each well, except for the blank control wells. Seal the plate again with sealing film and incubate at 37℃ for 30 min; ⑤ Second wash: Repeat the above washing steps, wash 5 times and pat dry; ⑥ Color development: Add 50 μL of color reagent A and 50 μL of color reagent B to each well, gently shake to mix, and incubate at 37℃ for 15 min in the dark; ⑦ Termination of reaction: Add 50 μL of stop solution to each well, gently shake to mix, at which point the liquid in the well changes from blue to yellow, terminating the color development reaction; ⑧ Reading: Immediately use an ELISA reader, zero the instrument with the blank control well, and measure the absorbance (OD value) of each well at a wavelength of 450 nm, and record the measurement results; ⑨ Calculate the content: Plot a standard curve based on the OD value of the standard, substitute the OD values ​​of each group of samples, and calculate the specific content of T4 and TSH in the serum of mice in each group.

[0090] (5.2.5) Histopathological examination: The fixed thyroid tissue was dehydrated in a gradient, embedded in paraffin, and serially sectioned; HE staining and Cleaved Caspase-3 (CC3) immunohistochemical staining were performed respectively; the thyroid follicular structure, epithelial cell status and CC3 positive cell ratio were observed under an optical microscope.

[0091] (5.3) Experimental Results

[0092] Experimental results are as follows Figures 20-23As shown, ELISA results indicated that the Ctrl group had the highest serum T4 level and the lowest TSH level. The NDV and Ruxo groups showed slightly lower T4 levels and slightly higher TSH levels compared to the Ctrl group. The Ruxo+NDV combination group exhibited the most significant decrease in T4 and increase in TSH among the four groups, suggesting that the combined intervention of NDV and ruxotinib synergistically reduces serum T4 and increases TSH, demonstrating a significantly better regulatory effect on thyroid hormone levels than single-drug or single-virus treatments. Immunohistochemical results showed that the thyroid follicle structure in the Ctrl group was regular, and the apoptosis marker CC3 was only expressed at a basal level. The NDV group increased CC3 expression, effectively inhibiting thyroid cell proliferation and inducing apoptosis. The CC3 expression level in the Ruxo monotherapy group was similar to that in the Ctrl group, while the Ruxo+NDV group significantly upregulated CC3 compared to the NDV monotherapy group, demonstrating a significant synergistic effect in inhibiting cell proliferation and promoting apoptosis.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of NDV in the preparation of drugs for treating hyperthyroidism.

2. Application of NDV in the preparation of drugs that enhance the anti-hyperthyroidism efficacy of JAK inhibitors.

3. The application according to claim 2, characterized in that, The NDV is a non-virulent or attenuated Newcastle disease virus strain.

4. The application according to claim 2, characterized in that, The JAK inhibitors include AG490 or ruxolitinib.

5. A drug for treating hyperthyroidism, characterized in that, The drug includes NDV; The final concentration of NDV in the drug is 1~100 MOI.

6. The drug according to claim 5, characterized in that, The drug also includes JAK inhibitors; The JAK inhibitors include AG490 or ruxolitinib.

7. The drug according to claim 6, characterized in that, The final concentration of AG490 in the drug is 1~100 nM.

8. The drug according to claim 6, characterized in that, The final concentration of ruxolitinib in the drug is 0.37~9.9 nM.

9. The drug according to any one of claims 5 to 8, characterized in that, The dosage form of the drug includes injections.

10. The medicament according to claim 9, characterized in that, The drug includes pharmaceutically acceptable excipients.

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