Ivermectin action target protein and application thereof

Abstract

The invention relates to an ivermectin action target protein and application thereof, and belongs to the technical field of biomedicine. The invention provides a drug action target protein for ovarian cancer, discloses a mechanism network of the anti-ovarian cancer effect of ivermectin and the intervention effect of ivermectin on an ovarian cancer energy metabolism pathway through experiments, and evaluates the in-vitro / in-vivo anti-ovarian cancer effect of ivermectin and the regulation effect of ivermectin on energy metabolism. The target protein of the ivermectin acting on the ovarian cancer cells is determined through a'click chemistry 'technology, a fluorescent probe technology and a target hooking mode, and then the target protein of the ivermectin acting on the ovarian cancer cells is obtained through further analysis and can be used for prediction, preclinical prevention and individualized medication of the ovarian cancer. The development of clinical medication for ovarian cancer under a medical framework is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an ivermectin target protein and applications thereof. Background Art

[0002] Ovarian cancer is the malignant tumor with the highest mortality rate in the female reproductive system. It is characterized by a low rate of early diagnosis, short-term recurrence, and high resistance to chemotherapy. Although standardized first-line treatment has prolonged progression-free survival in some patients, recurrence is inevitable, and effective second-line treatment options are still lacking in clinical practice. Existing drugs are no longer effective in treating ovarian cancer, making it a difficult problem in clinical treatment. There is an urgent need for new therapeutic agents to supplement established standard chemotherapy.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide an ivermectin target protein and its application, so as to solve the problem of lack of drugs for treating ovarian cancer in the prior art.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a drug target protein for ovarian cancer, wherein the drug target protein includes one or more of SERPINA, IGHG1, IGHA1, FGA, FGG, PDIA1, IGLC3, G22P1, RAC1, PSMA2, YWHAB, SFN, SHMT2, RPL28, IMA1, ARHGDIA, PSMA6, RAB10, PSME3, RPS14, RPL23A, TARDBP, TRAP2, CNN2, ZC3H13, TGFBRAP1 and HINT1.

[0007] The present invention provides the use of the drug target protein as an ovarian cancer disease marker.

[0008] The present invention provides the use of a reagent for detecting the drug target protein in preparing a kit for ovarian cancer diagnosis.

[0009] The present invention provides the use of a reagent for detecting the drug target protein in preparing a kit for evaluating the prognosis of ovarian cancer.

[0010] The present invention provides the application of the drug target protein as a screening target in screening drugs for treating ovarian cancer.

[0011] The present invention provides use of ivermectin in preparing a medicine for treating ovarian cancer.

[0012] Preferably, the dosage of ivermectin is 8-12 mg / kg.

[0013] Preferably, the ivermectin regulates the energy metabolism of ovarian cancer cells by acting on the drug target protein, thereby inhibiting the proliferation of ovarian cancer cells.

[0014] Preferably, the drug target protein is the drug target protein according to claim 1.

[0015] The present invention has the following technical effects and advantages:

[0016] The present invention establishes the protein change spectrum of ivermectin in vivo treatment of ovarian cancer, reveals the mechanism network of ivermectin's anti-ovarian cancer effect, and analyzes the data of ovarian cancer cells TOV-21G treated with ivermectin, revealing the intervention effect of ivermectin on the energy metabolism pathway of ovarian cancer.

[0017] The present invention evaluates the anti-ovarian cancer effects of ivermectin in vitro and in vivo and its regulatory effects on energy metabolism, and determines the target protein of ivermectin on ovarian cancer cells through "click chemistry" technology, fluorescent probe technology and target fishing. The target protein of ivermectin is then further analyzed and obtained, which can be used for the prediction, preclinical prevention and personalized medication of ovarian cancer, and promote the development of clinical medication for ovarian cancer within the medical framework. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a simplified structural diagram of ivermectin;

[0019] Figure 2 Figure 2 is the cell activity test results of different treatment groups;

[0020] Figure 3 Figure 2 is the result of cancer cell migration in different treatment groups;

[0021] Figure 4 Figure 2 shows the invasion results of cancer cells in different treatment groups;

[0022] Figure 5 This is the flow chart of the in vivo anti-ovarian cancer experiment of ivermectin;

[0023] Figure 6 This is a graph showing the changes in body weight, tumor weight, and tumor volume of mice in different treatment groups;

[0024] Figure 7 HE staining images of heart, liver, spleen, lung, kidney and tumor tissues of mice in different treatment groups;

[0025] Figure 8 Figure 2 shows the Tunel staining and analysis results of tumor tissues in mice in different treatment groups;

[0026] Figure 9 Figure 2 shows the results of Hoechst33258 staining of tumor tissues of mice in different treatment groups;

[0027] Figure 10 The results of multiple immunofluorescence staining of BAX and Bcl-2 proteins in ovarian cancer cells of mice in different treatment groups;

[0028] Figure 11 The results of multiple immunofluorescence staining of Cyt-2, Caspase9 and Caspase3 proteins in ovarian cancer cells of mice in different treatment groups;

[0029] Figure 12 This is a graph showing the glycolysis analysis of ovarian cancer cells in different treatment groups;

[0030] Figure 13 This is the detection diagram of mitochondrial respiration in tumor tissues of mice in different treatment groups;

[0031] Figure 14 This is a curve chart of the RPPA standard test results;

[0032] Figure 15 Unsupervised hierarchical clustering heatmap of RPPA data;

[0033] Figure 16 Semi-supervised hierarchical clustering heatmap of RPPA data;

[0034] Figure 17 Target annotation result diagram for RPPA data;

[0035] Figure 18 This is the PCA analysis diagram of RPPA data;

[0036] Figure 19 Volcano plot of differentially expressed proteins in TOV-21G cells treated with ivermectin;

[0037] Figure 20 This is a heat map of the whole protein of TOV-21G cells treated with ivermectin;

[0038] Figure 21 This is the cluster diagram of the top 10 differentially expressed proteins in TOV-21G cells treated with ivermectin;

[0039] Figure 22 GO analysis of differentially expressed proteins detected by RPPA;

[0040] Figure 23 This is a KEGG scatter plot with significant enrichment;

[0041] Figure 24 This is a diagram of the AMPK signaling pathway;

[0042] Figure 25 Figure 1 is a diagram of the autophagy signaling pathway;

[0043] Figure 26 The mitochondrial morphology of human ovarian cancer cell TOV-21G observed by transmission electron microscopy;

[0044] Figure 27 The mitochondrial morphology of mouse ovarian cancer tissue was observed by transmission electron microscopy;

[0045] Figure 28 Experimental flow chart for screening ivermectin target proteins using click chemistry technology;

[0046] Figure 29 is the HPLC-MS spectrum of ivermectin with molecular probe;

[0047] Figure 30 Ivermectin with molecular probes 1 H-NMR spectrum;

[0048] Figure 31 The in-gel fluorescence experiment results are shown for the optimal protein binding concentration and the optimal competitive concentration of the probe;

[0049] Figure 32 Survival analysis results of SERPINA, IGKC, IGHG1, and IGHA1 molecular proteins;

[0050] Figure 33 Survival analysis results of FGA, FGG, PDIA1, and IGH@molecular proteins;

[0051] Figure 34 Survival analysis results of G22P1, RAC1, WARS, and EEF1B2 molecular proteins;

[0052] Figure 35 Survival analysis results of PSMA2, YWHAB, SFN, and SHMT2 molecular proteins;

[0053] Figure 36 Survival analysis results of RPL28, KPNA2, ARHGDIA, and PSMA6 molecular proteins;

[0054] Figure 37 Survival analysis results of RAB10, PSME3, RPS14, and RPL23A molecular proteins;

[0055] Figure 38 Survival analysis results of TARDBP, TRAP2, TRIM28, and CNN2 molecular proteins;

[0056] Figure 39 Survival analysis results of ZC3H13, SERPINB12, TGFBRAP1, and HINT1 molecular proteins;

[0057] Figure 40 This is the molecular docking diagram of ACE2 and ivermectin;

[0058] Figure 41 This is the molecular docking diagram of TMPRSS2 and ivermectin;

[0059] Figure 42 This is the molecular docking diagram of MPro and ivermectin;

[0060] Figure 43 This is the molecular docking diagram of SERPINA and ivermectin;

[0061] Figure 44 This is the molecular docking diagram of IGHG1 and ivermectin;

[0062] Figure 45 This is the molecular docking diagram of IGHA1 and ivermectin;

[0063] Figure 46 This is the molecular docking diagram of FGA and ivermectin;

[0064] Figure 47 This is the molecular docking diagram of FGG and ivermectin;

[0065] Figure 48 This is the molecular docking diagram of PDIA1 and ivermectin;

[0066] Figure 49 Molecular docking diagram of (IGLC3)IGL@ and ivermectin;

[0067] Figure 50 This is the molecular docking diagram of (XRCC6)G22P1 and ivermectin;

[0068] Figure 51 This is the molecular docking diagram of RAC1 and ivermectin;

[0069] Figure 52 Molecular docking diagram of (PSA2)PSMA2 and ivermectin;

[0070] Figure 53 This is the molecular docking diagram of (1433B)YWHAB and ivermectin;

[0071] Figure 54 This is the molecular docking diagram of (1433S)SFN and ivermectin;

[0072] Figure 55This is the molecular docking diagram of (GLYM)SHMT2 and ivermectin;

[0073] Figure 56 This is the molecular docking diagram of (RL28)RPL28 and ivermectin;

[0074] Figure 57 This is the molecular docking diagram of (IMA1)KPNA2 and ivermectin;

[0075] Figure 58 This is the molecular docking diagram of (GDIR1)ARHGDIA and ivermectin;

[0076] Figure 59 Molecular docking diagram of (PSA6)PSMA6 and ivermectin;

[0077] Figure 60 This is the molecular docking diagram of RAB10 and ivermectin;

[0078] Figure 61 This is the molecular docking diagram of PSME3 and ivermectin;

[0079] Figure 62 Molecular docking diagram of (RS14)RPS14 and ivermectin;

[0080] Figure 63 This is the molecular docking diagram of (RL23A) RPL23A and ivermectin;

[0081] Figure 64 This is the molecular docking diagram of (TADBP)TARDBP and ivermectin;

[0082] Figure 65 This is the molecular docking diagram of (PSMD2)TRAP2 and ivermectin;

[0083] Figure 66 This is the molecular docking diagram of CNN2 and ivermectin;

[0084] Figure 67 This is the molecular docking diagram of (ZC3HD)ZC3H13 and ivermectin;

[0085] Figure 68 This is the molecular docking diagram of (TRAP1)TGFBRAP1 and ivermectin;

[0086] Figure 69 This is the molecular docking diagram of HINT1 and ivermectin. DETAILED DESCRIPTION

[0087] The technical solutions provided by the present invention are 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.

[0088] Example 1: Anti-ovarian cancer effect of ivermectin in vitro

[0089] 1. Materials and Methods

[0090] 1.1 Materials and Reagents

[0091] Human ovarian cell line (TOV-21G) was obtained from Wuhan Punosai Life Science Co., Ltd. and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin G / streptomycin (Gibco) at 37°C and 5% CO2, with the medium refreshed every 2 days. Ivermectin was provided by Beijing Solaibao Technology Co., Ltd., batch number A1107C023. The simplified structure of ivermectin is shown in Figure 2. Figure 1 Cisplatin (DDP) was provided by Qilu Pharmaceutical Co., Ltd., batch number: 3b0044b03. All other chemical reagents were of chromatographic grade.

[0092] Experimental groups: normal group, ivermectin low-dose group (5μM) and ivermectin high-dose group (10μM).

[0093] 1.2 Cell viability test

[0094] The CCK8 method was used to determine cell viability, and TOV-21G cells in the logarithmic growth phase and in good growth condition were selected for the experiment.

[0095] The experimental method is as follows: qualified cells are taken out, placed on a clean bench, rinsed 2-3 times with PBS, and the washing solution is discarded; digested with trypsin, centrifuged at 1300×g for 5 minutes at room temperature, and the supernatant is discarded; 1 mL of Gibco RPMI 1640 medium containing 5% fetal bovine serum is added to the centrifuge tube, blown evenly, mixed well, and the cells are counted; after counting, the cells are diluted proportionally to 5.0×10 7 / L, 100 μL was inoculated into each well of a 96-well plate, keeping the number of cells in each well unchanged; the inoculated cells were cultured in a CO2 incubator overnight until the cells adhered to the 96-well plate; they were treated with ivermectin for 24 hours, 10 μL of a 10% CCK8 culture medium mixed solution was added to each well, and the plates were placed in a CO2 incubator and cultured for another 2 hours; after removing the culture medium, the absorbance was measured at 450 nm using a microplate reader to calculate the survival rate of ovarian cancer cells. The normal group was treated with sterile water, the low-dose ivermectin group was treated with a 5 μM ivermectin aqueous solution, and the high-dose ivermectin group was treated with a 10 μM ivermectin aqueous solution. The cell activity test results of the different treatment groups are shown in Figure 2. Figure 2As shown, Figure A shows the microscopic observation of the ivermectin high-dose group and the normal group from 0 to 24 hours, and Figure B shows the changing trend of the cell viability of TOV-21G cells in the ivermectin high-dose group over time.

[0096] The results showed that 10 μM ivermectin could reduce the survival rate of TOV-21G cells to 65% in 4 hours and below 50% in 12 hours, showing a significant anti-ovarian proliferation effect.

[0097] 1.3 Cell migration assay

[0098] The cell chamber is placed in the corresponding culture plate. The inner chamber is called the upper chamber, and the inner chamber of the culture plate is called the lower chamber. The upper culture fluid is contained in the upper chamber, and the lower culture fluid is contained in the lower chamber. The upper and lower culture fluids are separated by a polycarbonate membrane with a pore size of 8.0 μm. The cells are inoculated in the upper chamber. Since the polycarbonate membrane is permeable, the components in the lower culture fluid can affect the cells in the upper chamber, so that the effects of the components in the lower culture fluid on cell growth, movement, etc. can be studied. In the cell migration and invasion experiment, tumor cells are inoculated in the upper chamber, and FBS or certain specific chemokines are added to the lower chamber. The tumor cells will move to the lower chamber with high nutritional content, thereby passing through one side of the porous membrane and adhering to the other side of the porous membrane. Counting the number of cells can reflect the migration or invasion ability of the tumor cells (the round transparent holes are micropores).

[0099] The specific methods of cell migration and invasion experiments are as follows:

[0100] (1) Place a 1.5 mL EP tube, pipette tip, and cell chamber (which has been placed in the well plate) on ice for pre-cooling.

[0101] (2) Dilute the ECM gel (EMC gel) to serum-free culture medium at a mass ratio of 1:7.5 on ice to prepare the working solution.

[0102] (3) Cut off 3 μm of the pipette tip, then draw 40 μL / well of the working solution on ice and gently add it to the upper chamber of the cell chamber. Move the pipette tip slowly while adding to ensure that the liquid is spread evenly on the bottom.

[0103] (4) Place the cell chamber in a 37°C incubator for 15 minutes to allow the gel to solidify.

[0104] (5) The cultured TOV-21G cells were digested, centrifuged, and counted, and the cells were counted according to the ratio of 2.5×10 4 The cells were diluted with serum-free medium to a concentration of 1:1 / mL to prepare a cell suspension.

[0105] (6) Add 200 μL of cell suspension to each well into the upper chamber, and add 500 μL of 10% FBS + culture medium into the lower chamber, and place in a 37°C incubator for culture.

[0106] (7) After several hours, remove the upper chamber, remove excess liquid, and wash twice with PBS. Use a cotton swab to gently rotate in the upper chamber to absorb water and wipe off cells on the inner side of the membrane.

[0107] (8) Add crystal violet dye to the upper chamber and dye for 5 minutes. Recover the dye and rinse it slowly with running water. Use a cotton swab to gently rotate in the upper chamber again to absorb the water.

[0108] (9) Place a glass slide on an upright microscope, place the cell chamber upside down on it, and take a picture.

[0109] (10) Under 100x magnification, count the top, bottom, left, right, and middle of the membrane and take the average.

[0110] The results of cancer cell migration in different treatment groups are as follows Figure 3 As shown, Figure A shows the 100x field of view microscope observation of different treatment groups, and Figure B shows the migration rate of ovarian cancer cells in different treatment groups.

[0111] according to Figure 3 It can be seen that compared with the control group, the number of migrating cells in the low-dose ivermectin group and the high-dose ivermectin group was significantly reduced, and the difference was statistically significant (P<0.05). Compared with the low-dose ivermectin group, the number of migrating cells in the high-dose ivermectin group also gradually decreased, and the difference was statistically significant (P<0.05).

[0112] 1.4 Cell invasion assay

[0113] The specific steps of the experiment are:

[0114] (1) Thaw the ECM Gel stock solution in a 4°C refrigerator for 1 hour in advance and transfer it to an ice box before the experiment.

[0115] (2) Place the 1.5 mL EP tube, pipette tip, and cell chamber (which have been placed in the well plate) on ice for pre-cooling.

[0116] (3) Dilute the ECM gel (EMC gel) to serum-free culture medium at a mass ratio of 1:7.5 on ice to prepare the working solution.

[0117] (4) Cut off 3 μm of the pipette tip, then draw 40 μL / well of the working solution on ice and gently add it to the upper chamber of the cell chamber. Move the pipette tip slowly while adding to ensure that the liquid is spread evenly on the bottom.

[0118] (5) Place the cell chamber in a 37°C incubator for 15 minutes to allow the gel to solidify.

[0119] (6) The cultured TOV-21G cells were digested, centrifuged, and counted, and the cells were counted according to the ratio of 2.5×10 4The cells were diluted with serum-free medium to a concentration of 1:1 / mL to prepare a cell suspension.

[0120] (7) Add 200 μL of cell suspension to each well into the upper chamber, and add 500 μL of 10% FBS + culture medium into the lower chamber, and place in a 37°C incubator for culture.

[0121] (8) After several hours, remove the upper chamber, remove excess liquid, and wash twice with PBS. Use a cotton swab to gently rotate in the upper chamber to absorb water and wipe off the cells on the inner side of the membrane.

[0122] (9) Add crystal violet dye to the upper chamber and dye for 5 minutes. Recover the dye and rinse it slowly with running water. Use a cotton swab to gently rotate in the upper chamber again to absorb the water.

[0123] (10) Place a glass slide on an upright microscope, place the cell chamber upside down on it, and take a picture.

[0124] (11) Under 100x magnification, count the top, bottom, left, right, and middle of the membrane and take the average.

[0125] The invasion results of cancer cells in different treatment groups are as follows Figure 4 As shown, Figure A shows the 100x field of view microscope observation of different treatment groups, and Figure B shows the invasion rate of ovarian cancer cells in different treatment groups.

[0126] according to Figure 4 It can be seen that compared with the control group, the number of invasive cells in the low-dose ivermectin group and the high-dose ivermectin group was significantly reduced, and the difference was statistically significant (P<0.05). Compared with the low-dose ivermectin group, the number of invasive cells in the high-dose ivermectin group also gradually decreased, and the difference was statistically significant (P<0.05).

[0127] Example 2: Anti-ovarian cancer effect of ivermectin in vivo

[0128] 1. Establishment of tumor-bearing mouse model and drug administration method

[0129] Experimental mice were 6- to 8-week-old Balb / c-nude female mice, specific pathogen-free, weighing 18 ± 2 g, purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Certificate No. [Lu] 2022-0006). All experimental procedures were carried out in strict accordance with the "Regulations on the Management of Laboratory Animals" issued by the Laboratory Animal Ethics Committee of Shandong First Medical University.

[0130] The experimental process of ivermectin against ovarian cancer in vivo is as follows Figure 5All mice were provided with standard laboratory diet and water, maintained at 25 ± 2 °C, with a 12-h light / dark cycle, and acclimated for at least one week. Female Balb / c-nude mice were randomly divided into five groups of 10 mice each. A concentration of 1.0 × 10 7 A TOV-21G cell suspension with a concentration of 10 cells / mL was prepared. The TOV-21G cell suspension was inoculated subcutaneously into the scapula of mice (0.15 mL per mouse) to establish a tumor-bearing mouse model. The tumor-bearing mice were randomly divided into a model group (MG), a positive treatment group, and an ivermectin-treated group (TG). The TG group was divided into high- and low-dose groups. The tumor-free mice served as the control group (n=10 per group).

[0131] The positive treatment group was intraperitoneally injected with cisplatin (DDP) at a dose of 10 mg / kg. The model group mice were gavaged with an equal amount of normal saline. The ivermectin treatment group mice were gavaged with 5 mg / kg in the low-dose group and 10 mg / kg in the high-dose group. Ivermectin was fully ground with a mortar in a sterile kitchen and added with normal saline to make a suspension for administration. Each group of mice was treated once a day for 15 consecutive days. After 15 days of treatment, the tissue specimens of each group were stored at -80°C, and the changes in the weight, tumor weight, and tumor volume of the mice were statistically analyzed. The results of the changes in the weight, tumor weight, and tumor volume of the mice in different treatment groups are shown in the figure. Figure 6 As shown, Figure A shows the tumor images of mice in different treatment groups, Figure B shows the changes in tumor weights of mice in different treatment groups, Figure C shows the changes in tumor volumes of mice in different treatment groups, Figure D shows the changes in body weights of mice in different treatment groups, and Figure E shows microscope images of ovarian cancer in different treatment groups.

[0132] according to Figure 6 After 15 days of continuous administration, the tumor mass and volume in the positive, high-dose, and low-dose treatment groups were statistically significantly different from those in the model group (p < 0.01), with tumor inhibition rates of 64.29%, 41.07%, and 60.71%, respectively. Following mouse modeling, tumor volume in the model group increased rapidly over time, while that in the positive and ivermectin treatment groups increased more slowly. High-dose ivermectin and the positive treatment groups significantly inhibited tumor growth. Body weight remained unchanged in the high-dose ivermectin group, while the positive treatment group experienced a significant decrease. Tumor cells in the model group were oval or round, dense, with intact and clear tumor tissue architecture and healthy growth. In contrast, tumor cells in the ivermectin treatment group were dispersed, with prominent nuclear proliferation. Necrotic tumor tissue was pale pink and fragmented, and was observed in all treatment groups. Necrosis became more pronounced with increasing treatment doses.

[0133] 2. HE staining analysis of organs and tumors of mice in each group

[0134] HE staining analysis was performed on the heart, liver, spleen, lung, kidney and tumor tissues of each group of mice. The specific experimental steps are as follows:

[0135] (1) Dewax paraffin sections to water

[0136] Sequentially place the sections in xylene I and soak for 10 min - xylene II and soak for 10 min - anhydrous ethanol I and soak for 5 min - anhydrous ethanol II and soak for 5 min - 95% alcohol and soak for 5 min - 90% alcohol and soak for 5 min - 80% alcohol and soak for 5 min - 70% alcohol and wash with distilled water.

[0137] (2) Hematoxylin staining of cell nuclei

[0138] The sections were stained with Harris hematoxylin for 5 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia solution, and rinsed with running water.

[0139] (3) Eosin staining of cytoplasm

[0140] The sections were stained with eosin solution for 2 min.

[0141] (4) Dehydration and sealing

[0142] Soak the sections in 95% alcohol I for 5 min - 95% alcohol II for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min - xylene II for 5 min to dehydrate and make them transparent. Take the sections out of xylene and let them dry slightly, then seal them with neutral gum.

[0143] (5) Microscopic examination, image acquisition and analysis

[0144] HE staining of heart, liver, spleen, lung, kidney and tumor tissues of mice in different treatment groups Figure 7 As shown in the figure, NP represents normal mice, MG represents model group, DDP represents positive treatment group, 5 mg / kg represents low-dose ivermectin treatment group, and 10 mg / kg represents high-dose ivermectin treatment group.

[0145] according to Figure 7 It can be seen that ivermectin had no significant toxic side effects on the heart, liver, spleen, lungs, and kidneys of the mice in each group. Ivermectin intervention promoted tumor cell apoptosis. Compared with the model group, after ivermectin treatment, the number of brown-yellow areas in the nuclei of ovarian cancer cells increased significantly, and the cells were scattered. After ivermectin treatment, the apoptosis rate of ovarian cancer cells increased, confirming that ivermectin can induce cell apoptosis and has a significant anti-tumor effect in TOV-21G tumor-bearing mice. The anti-tumor effect of ivermectin is dose-dependent.

[0146] 3. Tunel Analysis

[0147] The specific method is as follows:

[0148] (1) Tumor tissues of mice in each group were obtained and fixed with freshly prepared 4% formaldehyde at room temperature overnight. 100 mL of 2×PBS with a pH value of 7.2 was added, and the fixative was filtered to obtain tumor tissues of mice in each group.

[0149] (2) The tumor tissues of each group of mice were dehydrated by incubation in a series of 50%, 70%, 80%, 95%, 100% ethanol and 100% xylene.

[0150] (3) Embed the tissue in paraffin, prepare 5 μm thick sections, and fix them on glass slides.

[0151] (4) Wash with gradient ethanol (100, 95, 90, 80, 70%) once, each time for 3 minutes.

[0152] (5) After rinsing with PBS twice, the tissue was treated with Proteinase K working solution (20 μg / mL) for 20 min.

[0153] (6) Add PBS, react at room temperature for 5 minutes, and rinse with PBS twice

[0154] (7) Prepare the Tunel reaction mixture. The treatment group was mixed with 50 μL of TdT and 450 μL of fluorescein-labeled dUTP solution, while the negative control group was only added with 50 μL of fluorescein-labeled dUTP solution. The positive control group was first added with 100 μL of DNase 1 and reacted at 15-25°C for 10 min. The subsequent steps were the same as the treatment group.

[0155] (8) After the slides are dry, use filter paper to carefully remove excess liquid around the sections, add 50 μL of TUNEL reaction mixture (only 50 μL of fluorescein-labeled dUTP solution is added to the negative control group), cover the specimen with a glass slide or sealing film, and react in a dark humid box at 37°C for 1 hour.

[0156] (9) Add to the washing and termination reaction buffer preheated at 37°C, incubate at 37°C for 30 min, and rinse with PBS three times.

[0157] (10) Add one drop of PBS and count apoptotic cells under a fluorescence microscope (excitation wavelength is 475 nm, detection wavelength is 540 nm).

[0158] (11) After the slide is dry, add 50 μL of DIG-POD to the dry specimen, cover with a slide or sealing film, and react in a dark box at 37°C for 30 min.

[0159] (12) Rinse with PBS three times; add 75 μL of DAB substrate to the tissue and react at 20°C for 10 min.

[0160] (13) Rinse with PBS three times; counterstain with hematoxylin or methyl green after taking a picture, and immediately rinse with tap water after a few seconds. Dehydrate with graded alcohol, clear with xylene, and mount with neutral gum.

[0161] (14) Add a drop of PBS or glycerol and observe apoptotic cells (200 to 500 cells in total) under a light microscope and take photos.

[0162] Tunel staining and analysis results of tumor tissues of mice in different treatment groups are shown in Figure 2. Figure 8 As shown, Figure A shows the Tunel staining images of tumor tissues of mice in each group, and Figure B shows the quantitative analysis of Tunel staining results.

[0163] according to Figure 8 It can be seen that both the positive treatment group and the high-dose ivermectin treatment group had a significant effect on promoting TOV-21G cell apoptosis, and the high-dose ivermectin treatment group had a more significant effect on apoptosis (P < 0.01). Compared with the low-dose ivermectin treatment group and the high-dose ivermectin treatment group, the pro-apoptotic effect increased with increasing dose.

[0164] 4. Hoechst 33258 Staining Analysis

[0165] Hoechst staining is a technique that uses the fluorescent dye Hoechst to stain the cell nucleus. Changes in the morphology of the cell nucleus are observed under a fluorescence microscope to determine whether the cell has undergone apoptosis. In normal cells, the cell nucleus shows a uniform staining effect; while in apoptotic cells, the cell nucleus shows characteristics of nuclear condensation and fragmentation. This staining method provides an intuitive and simple way to detect the process of cell apoptosis. Hoechst33258 staining was used to detect nuclear apoptosis in tumor tissues of each group of mice. The specific experimental steps are as follows:

[0166] (1) Take an ordinary clean cover glass and soak it in 70% ethanol for 5 minutes. Wash it three times with cell culture PBS solution and then wash it once with cell culture medium. Place the cover glass in a six-well plate and inoculate cells overnight to make it 70% full.

[0167] (2) After stimulating cells to undergo apoptosis, the culture medium was aspirated and 0.5 mL of fixative (4% paraformaldehyde) was added and fixed for 20 min.

[0168] (3) Remove the fixative solution and wash twice with PBS, 3 minutes each time, aspirate all the liquid, and shake the tube several times by hand during washing.

[0169] (4) Add 0.5 mL of Hoechst 33258 staining solution (concentration: 5 mg / L) and stain for 5 minutes. Shake the plate several times by hand during staining.

[0170] (5) Wash twice with PBS, 3 minutes each time.

[0171] (6) Add a drop of anti-fluorescence quenching sealing solution to the slide and cover it with the coverslip with cells so that the cells are in contact with the sealing solution.

[0172] (7) Allow to dry at room temperature, observe under a fluorescence microscope (detection wavelength 360 nm, reference wavelength 450 nm), and take random photos. Blue cell nuclei can be detected under a fluorescence microscope. The excitation wavelength is around 350 nm, and the emission wavelength is around 460 nm. Image-Pro plus 6.0 software is used for quantitative analysis of apoptotic cell nuclei.

[0173] The results of Hoechst33258 staining of tumor tissues of mice in different treatment groups are as follows Figure 9 As shown, Figure A shows the Hoechst33258 staining of ovarian cancer cells in each group of mice, and Figure B shows the quantitative analysis of apoptosis results of ovarian cancer cells in each group of mice.

[0174] according to Figure 9 It can be seen that a more obvious nuclear condensation phenomenon was observed in the high-dose ivermectin treatment group, indicating that ivermectin has a significant pro-apoptotic effect on TOV-21G cells.

[0175] 5. Multiplex Fluorescent Immunostaining Analysis

[0176] Multiplex immunofluorescence is an immunostaining technique based on tyramide signal amplification (TSA). Tyramide salts, catalyzed by horseradish peroxidase (HRP), form highly reactive tyramide free radicals. These free radicals covalently bind to electron-rich tyrosine residues surrounding tissue antigens, resulting in their deposition in large quantities. These covalently bound deposits are more stable than hydrogen-bonded primary and secondary antibodies. Microwave treatment of the eluent allows for easy elution of hydrogen-bonded primary and secondary antibodies, while covalently bound tyrosine fluorophores remain permanently deposited at antigenic sites. Finally, the fluorescent signal detected by the fluorophores carried by the tyramide molecules provides antigen information. The ability to elute primary and secondary antibodies without affecting the labeling of other primary and secondary antibodies or different fluorophores allows for multiple labeling using different primary antibodies from the same species on the same slide, enabling the acquisition of high signal-to-noise ratio images using multispectral imaging systems.

[0177] Multiple fluorescent immunostaining was performed on the ovarian cancer tissues of each group of mice to determine the expression of five proteins, namely BAX, Bcl-2, Cyt-2, Caspase9, and Caspase3. The specific steps are as follows:

[0178] (1) Dewaxing of sections: Place the paraffin sections in the following order: xylene I (20 min) - xylene II (20 min) - xylene III (20 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min), and then rinse with distilled water for 5 min.

[0179] (2) Antigen repair: Use an electric ceramic furnace to heat the slices for antigen repair. Place the prepared repair solution (Tris-EDTA buffer, pH = 9.0) in a beaker and boil it over high heat. Then place the dewaxed and hydrated tissue slices on a high-temperature plastic slice rack in the beaker. The liquid level should be above the sliced ​​tissue to a certain height. At this time, the timer starts. The repair time is 15 minutes. Do not let the tissue dry during this process (the repair solution must be sufficient). After the time is up, remove the beaker from the electric ceramic furnace panel and place it at room temperature for about 40 minutes to cool down. When the repair solution reaches room temperature, remove the slides and rinse them with PBS (pH = 7.4) three times, each time for 3 minutes (do not rinse towards the tissue during the rinsing process to avoid damaging the tissue).

[0180] (3) Blocking endogenous peroxidase: Add prepared 3% hydrogen peroxide dropwise to the sliced ​​tissue to block endogenous peroxidase, incubate at room temperature for 15 min, and rinse with PBS three times, each time for 3 min.

[0181] (4) Serum blocking: Wipe the slide dry with absorbent paper, draw circles around the tissue with an immunohistochemistry brush, add diluted normal goat serum, and block at room temperature for 30 minutes to reduce nonspecific staining.

[0182] (5) Add primary antibody: Wipe the liquid around the tissue on the slide with absorbent paper, draw a circle around the tissue with a marker pen, and then add the diluted primary antibody. If a negative control experiment is performed, add PBS to the tissue in the control group. After adding the primary antibody, incubate in a humidified chamber at 4°C overnight (15 hours).

[0183] (6) Add enzyme-labeled secondary antibody: Rinse the sections with PBS three times, 3 minutes each time, dry the sections with absorbent paper, and then add HRP-labeled goat anti-rabbit / mouse secondary antibody and incubate at 37°C for 30 minutes.

[0184] (7) Rinse the sections with PBS three times for 3 minutes each time. Dry the sections with absorbent paper and add TSA-Fluorescein (diluted with Amplification Dilution) and incubate at 37°C for 10 minutes.

[0185] (8) Microwave repair (removal of primary and secondary antibodies): Rinse the sections three times with PBS for 3 minutes each time. Soak the sections in 1×AR6 Buffer and microwave on high heat until boiling, then keep on medium heat for 5 minutes. Then cool naturally. When the repair solution cools to room temperature, remove the slides and rinse them three times with PBS (pH = 7.4) for 3 minutes each time (do not rinse directly on the tissue during the rinsing process to avoid damaging the tissue). Repeat steps 4-8 until all primary antibodies have been added.

[0186] (9) Nuclear restaining: Add DAPI and incubate in the dark for 5 minutes to stain the nucleus of the specimen. Rinse with PBST 4 times, each time for 5 minutes to wash away excess DAPI.

[0187] (10) Wipe the liquid on the slice with absorbent paper and seal the slice with sealing solution containing anti-fluorescence quenching agent.

[0188] (11) Microscopic examination and photography: The slices were imaged and analyzed using the PE Vectra automated multispectral histopathology quantitative analysis system.

[0189] Compared with traditional flow cytometry or immunohistochemistry, multiplex fluorescent immunostaining can increase the rate of discovering new biomarkers and therapeutic target pathways. This is because multiplex fluorescent immunostaining can define and characterize the tissue microenvironment from three dimensions: qualitative, quantitative, and localized, providing richer data dimensions and deeper analytical capabilities. The results of multiplex immunofluorescence staining of BAX and Bcl-2 proteins in mouse ovarian cancer cells under different treatment groups are shown in Figure 2. Figure 10 As shown in Figure 2, the results of multiple immunofluorescence staining of Cyt-2, Caspase9 and Caspase3 proteins in ovarian cancer cells of mice in different treatment groups are shown in Figure 2. Figure 11 shown.

[0190] according to Figure 10 and Figure 11 Compared with the model group, the high-dose ivermectin treatment group showed a significant upregulation of cytochrome C (Cyt-c), caspase-3, and caspase-9 expression. This suggests that ivermectin treatment activates the endogenous mitochondrial apoptosis pathway in tumor cells, thereby exerting an anti-tumor effect.

[0191] 6. Study on the inhibition of energy metabolism rate of ovarian cancer cells / tissues by ivermectin

[0192] The cellular glycolysis function was evaluated by comparing and analyzing multiple parameters including cellular basal glycolysis, maximum glycolysis capacity, glycolysis capacity, glycolysis reserve and total extracellular pH change, and the effect of ivermectin on the energy metabolism rate of ovarian cancer cells / tissues was determined. The results were as follows: Figure 12 and Figure 13 As shown, Figure 12Figure 2 shows the glycolysis analysis of ovarian cancer cells in different treatment groups. Figure A shows the glycolytic acid production efficiency of human ovarian cancer cells TOV-21G and human normal ovarian cells IOSE80. Figure B shows the real-time dynamics of glycolytic acid production efficiency of human ovarian cancer cells TOV-21G. Figure C shows the real-time efficiency of mitochondrial respiration oxygen consumption of human ovarian cancer cells TOV-21G and human normal ovarian cells IOSE80. Figure D shows the real-time dynamics of mitochondrial respiration oxygen consumption efficiency of human ovarian cancer cells TOV-21G. Figure 13 This is the mitochondrial respiration detection of mouse tumor tissue in different treatment groups. Figure A shows the mitochondrial respiration oxygen consumption efficiency of mouse tumor tissue, Figure B shows the real-time efficiency of mitochondrial respiration oxygen consumption of mouse tumor tissue, and Figure C shows the mitochondrial respiration consumption detection of mouse tumor tissue.

[0193] according to Figure 12 and Figure 13 Compared with normal human ovarian cells (IOSE80), ivermectin-treated human ovarian cancer cell lines TOV-21G showed a significant decrease in glycolytic capacity, as reflected in changes in total extracellular pH, maximum glycolytic capacity, glycolytic capacity, and glycolytic reserve. Compared with human ovarian cancer cell lines TOV-21G, ivermectin-treated human ovarian cancer cell lines TOV-21G showed a significant decrease in glycolytic capacity, as reflected in changes in total extracellular pH, basal glycolysis, and maximum glycolytic capacity. Glycolytic capacity measurements in TOV-21G and IOSE80 cells indicated that ivermectin inhibited cellular glycolytic function. Compared with human ovarian cancer cell lines TOV-21G, ivermectin-treated human ovarian cancer cell lines TOV-21G showed a significant decrease in basal respiration, complex II oxidative phosphorylation, maximum respiration, and non-mitochondrial oxygen consumption. Compared with normal human ovarian cells IOSE80, ivermectin-treated human ovarian cancer cell lines TOV-21G showed a decrease in basal respiration, complex II oxidative phosphorylation, maximal respiration, and non-mitochondrial oxygen consumption to some extent, but without significant differences. This suggests that ivermectin can inhibit mitochondrial respiration in tumor cells, thereby inhibiting tumor tissue growth. Ivermectin also exhibited no significant toxic side effects. This suggests that ivermectin significantly inhibits the rate of energy metabolism in ovarian cancer both in vitro and in vivo.

[0194] 7. Reverse Phase Protein Array (RPPA) Research

[0195] Reverse Phase Protein Array (RPPA) is a high-throughput proteomics technology used for quantitative analysis of proteins and their post-translational modification states. We used reverse phase protein array (RPPA) to study ovarian cancer cells. The experimental method is as follows:

[0196] (1) Sample preparation

[0197] 1) Incubate cells in a six-well culture plate / 90mm culture plate until the cell density reaches 80%. The number of cells is determined by cell size, cell growth rate, and experimental design (e.g., culture duration and method). The generally recommended cell density per well of a six-well culture plate is: 0.3-0.5×10 6 / 3mL, the concentration of 90mm culture dish is: 2~5×10 6 pcs / 10mL.

[0198] 2) Cell treatment was performed according to the experimental design.

[0199] 3) After washing the cells twice with PBS, be sure to aspirate any remaining liquid and add lysis buffer, 100-150 μL per well of a six-well plate or 300-500 μL per 90 mm culture plate, ensuring that the lysis buffer covers the entire surface.

[0200] 4) Place the plate on ice (horizontally) and incubate for 15-20 minutes, shaking every 5 minutes to evenly distribute the lysate.

[0201] 5) Scrape the cells and collect the cell lysate into a centrifuge tube. Centrifuge at 14,000 rpm (maximum speed) and 4°C for 10 min.

[0202] 6) Carefully collect the supernatant and discard the precipitate.

[0203] 7) Store at -80℃ until sample delivery.

[0204] (2) Sample pretreatment

[0205] First, tissues or cells were lysed and proteins were extracted using the standard RPPA sample preparation SOP. The sample concentration was then quantified using the BCA assay. The sample to be tested was diluted to 1.5 μg / μL using lysis buffer. Six groups of tumor cells from mice in the model group (MG) and six groups of tumor cells from mice in the high-dose ivermectin treatment group (TG) were collected for sample quantification QC. The sample quantification QC results are shown in Table 1:

[0206] Table 1 Sample quantitative QC results

[0207] sample 384-wellPlate 384-welldefinition ID Lysis buffer concentration (mg / mL) MG1 Plate 4 B3,F3,J3,N3,B15 20240617001001 2.22 MG2 Plate6 B3,F3,J3,N3,B15 20240617001002 1.83 MG3 Plate8 B3,F3,J3,N3,B15 20240617001003 1.95 MG4 Plate10 B3,F3,J3,N3,B15 20240617001004 1.92 MG5 Plate12 B3,F3,J3,N3,B15 20240617001005 1.96 MG6 Plate 14 B3,F3,J3,N3,B15 20240617001006 1.98 TG1 Plate16 B3,F3,J3,N3,B15 20240617001007 1.49 TG2 Plate18 B3,F3,J3,N3,B15 20240617001008 1.58 TG3 Plate20 B3,F3,J3,N3,B15 20240617001009 1.8 TG4 Plate22 B3,F3,J3,N3,B15 20240617001010 1.4 TG5 Plate 4 C3,G3,K3,O3,C15 20240617001011 1.55 TG6 Plate6 C3,G3,K3,O3,C15 20240617001012 1.47

[0208] (3) Sample gradient dilution

[0209] Using a Tecan Fluent 480 / 780 liquid workstation, the sample (tissue or cell) lysate was serially diluted (a total of 5 dilutions: 1 (undiluted), 1 / 2, 1 / 4, 1 / 8, and 1 / 16).

[0210] (4) Chip printing

[0211] The samples were printed and fixed onto nitrocellulose membrane chips using a Quanterix 2470 Arrayer in 11×11 subarrays.

[0212] (5) Antibody incubation and color development

[0213] The protein chip was incubated with the primary antibody to be detected and the corresponding secondary antibody on an Agilent AutostainerLink 48 automatic stainer, and the signal was amplified using tyramide signal amplification method and the color was developed using DAB colorimetric reaction.

[0214] (6) Chip scanning

[0215] The colorimetric chip was imaged on a Huron TissueScope LE120 scanner to generate 16-bit tiff raw images (each chip contained 5808 data points).

[0216] (7) Original data acquisition

[0217] MicroVigene was used to identify and digitize the sample points in the tiff image and the raw data were output.

[0218] (8) The acquired data were integrated and analyzed, including basic analyses such as heat maps, target annotations, and PCA plots, and differential expression analyses such as volcano plots, boxplots, and KEGG analysis.

[0219] The above method was used to perform reverse phase protein array (RPPA) studies on TOV-21G cells treated with ivermectin.

[0220] A. RPPABCA total protein content test results

[0221] ①. Sample processing and detection methods

[0222] The BCA assay was used. A linear relationship was fitted between the standard concentration and its average absorbance. The average sample absorbance was substituted into this linear relationship to obtain the sample test solution concentration. Sample concentration = sample test solution concentration × 15 (dilution factor). The standard test results are shown in Table 2.

[0223] Table 2 Standard test results

[0224]

[0225] ②. Establishment of annotation curve

[0226] A standard curve was constructed based on the test results of the standard product. The curve of the RPPA standard product test results is shown in the figure below. Figure 14 shown.

[0227] ③.Test results

[0228] The results of protein content detection of tumor cells in 6 groups of model groups (MG) mice and 6 groups of ivermectin high-dose treatment groups (TG) mice are shown in Table 3.

[0229] Table 3 Results of tumor cell protein content detection in different treatment groups

[0230]

[0231]

[0232] According to Table 3, all samples were rated as qualified samples.

[0233] B. Data Quality Control (QC) Results

[0234] ①. Chromatography quality control

[0235] Staining quality control reflects the reliability of the antibody data in this round of RPPASet, and is divided into four levels: Excellent, Good, Acceptable, and Cautious. Comprehensive quality control analysis showed that 86.7% of the antibody staining results were rated Excellent or Good.

[0236] ②. Sample quality control

[0237] The correction factor CF1 is used to correct for sample loading. If CF1 is less than 0.25 or greater than 2.5, it is considered abnormal, indicating that the sample concentration is much lower or higher than other samples.

[0238] C. Data Analysis Results

[0239] ①. Unsupervised hierarchical clustering heat map analysis (both antibodies and samples are unsupervised)

[0240] According to the result data analysis, the unsupervised hierarchical clustering heat map of RPPA data is as follows Figure 15 As shown in the figure, the semi-supervised hierarchical clustering heat map of RPPA data is shown in the figure. Figure 16 As shown, the samples are arranged according to grouping information.

[0241] ②. Target Annotation

[0242] The KEGG pathway to which each target belongs and whether it is an approved drug target are annotated. The drug target information comes from DrugBank (Note: the row labels of the heat map are the annotations of the KEGG pathway or approved drug target, and the column labels are the target names). The target order is the same as that of the unsupervised and semi-supervised heat maps. The target annotation results of RPPA data are as follows: Figure 16As shown in the figure, the "WithApprovedDrug" row label (blue area) indicates whether the target has an approved drug target. All other areas are KEGG secondary pathways (red area). Red in the heatmap indicates that the target belongs to the current pathway or is an approved drug target, while white indicates that it is not. Gray indicates that the annotation information for the current target is unknown.

[0243] After data analysis, 384 proteins were detected after ivermectin treatment of TOV-21G cells. The abundance expression results of 384 proteins after ivermectin treatment of TOV-21G cells are shown in Table 4.

[0244] Table 4 Abundance expression results of 384 proteins after ivermectin treatment of TOV-21G cells

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] ④PCA analysis results

[0258] Perform principal component analysis (PCA) based on the grouping information and display the information of the first two principal components. The PCA analysis results of RPPA data are as follows: Figure 18 shown.

[0259] ⑤ Differential expression analysis

[0260] A volcano plot analysis was performed on the proteins with significant differences among the 384 proteins after ivermectin treatment of TOV-21G cells. The multiples were converted to Log2, and the two vertical lines of -1 and 1 in the volcano plot were on both sides of the differential expression. By the same token, the P value was converted to -log10, and -log10(0.05) was approximately equal to 1.30103, which is the horizontal dotted line in the volcano plot. The value below the dotted line is not significant. The smaller the P value, the more significant it is. Therefore, after we perform -log10 (P value) conversion, the larger the conversion value, the more significant the difference. The cutoff of Log2FC is 1, and the cutoff value of -log10 (P value) is 1.3. The volcano plot of differentially expressed proteins in TOV-21G cells treated with ivermectin is shown below. Figure 19 shown.

[0261] ⑥Heat map of TOV-21G cells treated with ivermectin Figure 20 As shown, the cluster diagram of the top 10 differentially expressed proteins in TOV-21G cells treated with ivermectin is shown in Figure 21 shown.

[0262] ⑦ Perform GO analysis on the data. The GO analysis diagram of the differential proteins detected by RPPA is as follows: Figure 22 The horizontal axis of the figure represents GeneRatio (the ratio of the total number of genes belonging to this GO term in the differentially expressed genes to the total number of differentially expressed genes). A larger value indicates a higher degree of enrichment. The vertical axis displays the top 20 GOTerms based on the -Log10 (P value) ranking information. Panel A represents the biological process (BP) enrichment network diagram, Panel B represents the cellular component (CC) enrichment network diagram, and Panel C represents the molecular function (MF) enrichment network diagram.

[0263] ⑧Significantly enriched KEGG scatter plot as shown Figure 23 As shown, according to Figure 23 It can be seen that the protein target list of the Cancer Signaling Pano-Profiler Cancer Signaling Panorama Analysis Panel covers many KEGG pathway genes. Here, 6 of the most covered and important KEGG pathways are selected for display. The log2FC (Fold Change takes the logarithm with base 2) value of the current inter-group comparison will be marked on each pathway diagram, represented by color. The grouping information involved in the calculation of log2FC is located in the selected group name. For example, if the group is Treatment_VS_Control, the calculation of log2FC is log2 (Treatment / Control). The results show that 26 signaling pathways including the AMPK signaling pathway and the autophagy signaling pathway were detected, among which the AMPK signaling pathway is as follows. Figure 24 As shown in Figure 25The KEGG enrichment analysis data are shown in Table 5.

[0264] Table 5 KEGG enrichment analysis data

[0265]

[0266]

[0267]

[0268]

[0269] Based on the above experimental results, RPPA analysis of ovarian cancer cells TOV-21G in the ivermectin treatment group and the blank control group in vitro confirmed that ivermectin plays a major role through the autophagy pathway.

[0270] Example 3: Transmission electron microscopy observation of mitochondrial autophagy in ovarian cancer cells

[0271] (1) Experimental equipment and reagents

[0272] 1) Experimental equipment:

[0273] Transmission electron microscope (Hitachi, HT7800 / HT7700); 150-mesh Fanghua membrane copper grid (Hyde Chuangye (Beijing) Biotechnology Co., Ltd., FF150).

[0274] 2) Key reagents: Electron microscopy fixative (Servicebio, G1102); anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 100092183); acetone (Sinopharm Chemical Reagent Co., Ltd., 10000418); osmium oxide (Ted Pella Inc., 18456); uranyl acetate (SPI, 02624-AB); trisodium citrate (Sinopharm Chemical Reagent Co., Ltd., 10019408); and lead nitrate (Sigma, 203580).

[0275] (2) Treatment of TOV-21G ovarian cancer cells

[0276] TOV-21G cells were removed from liquid nitrogen, cultured after recovery, and after 2-3 passages, placed in 6-well plates for culture. Equal amounts of DMSO and normal saline were added to the MG group, and 10 μM (with DMSO for solubility) was added to the TG group (high-dose ivermectin treatment group). The cells were placed in a 5% CO2 incubator and removed after 12 h.

[0277] Discard the cell culture medium and wash repeatedly with PBS 2-3 times. Carefully scrape the adherent cells with a cell scraper. Add PBS and gently pipette to mix. Centrifuge at 4°C, 1000 × g, for 5 minutes. Discard the supernatant and place the cells at the bottom in electron microscopy fixative. Store in a refrigerator at 4°C until ready to use.

[0278] (3) Operation steps of transmission electron microscope

[0279] 1) Power-on Preparation: Turn on the main unit and wait for the unit to reach the required vacuum level, which takes approximately half an hour. Check that the cooling water and main unit power are on. If there are any malfunctions during power-on, the fault will be displayed on the lower side of the LCD screen. Check the status of the transmission electron microscope: If any abnormalities are detected in the vacuum or high-voltage status of the transmission electron microscope, discontinue use and contact a technician immediately during normal operating hours.

[0280] 2) Filling the Dewar flask with liquid nitrogen: Wear gloves and carefully pour the liquid nitrogen into the Dewar flask, but do not fill it up. Place the Dewar flask on a stand and ensure that there is enough liquid nitrogen in the Dewar flask, so fill it up every 3 to 4 hours.

[0281] 3) Loading the sample: Select the sample rod, remove the front sleeve, and check that the sample rod and the fixture are clean and dry.

[0282] 4) Inserting the sample rod: Hold the end of the sample rod horizontally, aligning the positioning pin on the sample rod with the slit on the sample stage. Slowly insert the sample rod until it stops. A red light on the TEM stage will illuminate, and the mechanical pump will begin pre-evacuating the sample chamber airlock. After approximately 3 minutes, the red light on the TEM stage will go out. Rotate the sample rod counterclockwise until it stops. Then, firmly grasp the end of the sample rod and allow the vacuum to pull it slowly into the TEM.

[0283] 5) Add filament current: When the column vacuum value drops below 20, click the Filament indicator. The filament will automatically increase to the preset value, and the Filament indicator will turn yellow. Note the Emission and other values ​​before and after adding the filament.

[0284] Transmission electron microscope operation method:

[0285] 6) Finding the light: After the filament is turned on and the filament current is emitted, the electron beam should be visible on the screen. If no electron beam is visible after turning on the filament, perform the following steps: Lower the magnification to approximately 10K, move the trackball, and remove all apertures.

[0286] 7) Filament Image Adjustment: At ×40K magnification, use beamshift to move the beam spot to the center of the screen. Slowly reduce the filament current and use the BRIGHTNESS knob to minimize the beam spot. As the filament current decreases, a filament image should appear. Adjust the beam spot to SPOTSIZE 5, use beamshift to move it to the center of the screen, switch to SPOTSIZE 1, and use gunshift to move the offset beam spot back to the center of the screen. Repeat this process until the beam spot is essentially stationary when switching between SPOTSIZEs 1 and 5.

[0287] 8) Condenser anti-astigmatism: Observe the shape of each beam spot with a transmission electron microscope. If the beam spot is found to be elliptical, use the CONDSTIG key to adjust the beam spot to a circular shape.

[0288] 9) Add condenser iris: The red dot on the condenser iris indicates exit, and the size of the remaining dots indicates the size of the currently added iris. Add iris clockwise to the appropriate aperture, usually No. 1 or No. 2. After adding, use beamshift to move the light spot to the center of the screen. At this time, turn BRIGHTNESS clockwise to enlarge the light spot to the same size as the screen. If the iris is not added correctly, the light spot will not cover the screen evenly, that is, the light spot will not be enlarged concentrically with BRIGHTNESS. At this time, adjust the two knobs on the front and right side of the iris to make the center of the light spot concentric with the screen.

[0289] 10) Sample height adjustment: Find a sample, press IMAGEWOBBLEX or Y, observe the vibration of the transmission electron microscope image, and adjust the Z key until the image vibration amplitude is minimized.

[0290] 11) Voltage center adjustment: Find a sample and move it to the black dot in the middle of the screen.

[0291] Fine focus: Adjust the OBJFOCUS button to move the sample to be observed by the transmission electron microscope to the orthogonal position, and confirm the orthogonality through under-intersection, orthogonality, and over-intersection phenomena.

[0292] 12) Remove the sample rod: Reverse the steps for inserting the sample rod and carefully remove the sample from the sample rod, making sure that the sample does not damage or contaminate other parts.

[0293] Transmission electron microscopy observation of mitochondrial morphology of human ovarian cancer cell TOV-21G Figure 26 As shown in the figure, A represents the electron microscopic observation pictures of ovarian cancer cells with different treatments, and the magnification from left to right is 2500X, 8000X and 20000X, respectively; B represents the quantitative analysis of mitochondrial autophagy, MG represents the normal saline control group, TG represents the ivermectin treatment group, *** represents P < 0.001 compared with the model group MG.

[0294] according to Figure 26 It can be seen that ivermectin has a significant effect on the morphology and function of mitochondria in ovarian cancer cells TOV-21G. It can be clearly seen that the mitochondria in the MG group are oval or rod-shaped, with a clear double membrane structure. The outer membrane is smooth, and the inner membrane folds inward to form cristae. The cristae increase the surface area of ​​the inner membrane and contribute to energy production. The density of the internal matrix is ​​uniform, and some electron-dense particles can be seen. These are enzyme complexes involved in oxidative phosphorylation. The mitochondria in the TG group are wrapped by autophagosomes and surrounded by additional membrane structures. In terms of morphological changes, the mitochondria in the TG group appeared swollen or deformed, with irregular shapes. The integrity of the inner and outer membranes was destroyed, and the cristae became blurred or disappeared. The contents of the mitochondria were degraded, manifested as changes in electron density, vacuolation, or uneven contents. The mitochondria in the TG group showed significant autophagy, in sharp contrast to the normal mitochondria in the MG group.

[0295] Example 4: Transmission electron microscopy observation of mitochondrial autophagy in ovarian cancer tissue

[0296] In order to detect the effects of ivermectin on mitochondria in ovarian cancer tissues of experimental animals before and after treatment, transmission electron microscopy was used.

[0297] (1) Experimental equipment and reagents

[0298] 1.1 Experimental equipment: ultrathin microtome (Leica, Leica UC7), diamond microtome (Daitome, Ultra45°), transmission electron microscope (hitachi, HT7800 / HT7700).

[0299] 1.2 Main experimental reagents: electron microscopy fixative (Servicebio, G1102), anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., 100092183), acetone (Sinopharm Chemical Reagent Co., Ltd., 10000418), 812 (embedding medium, SPI90529-77-4), osmium phosphate (Ted Pella Inc, 18456), uranyl acetate (SPI, 02624-AB), trisodium citrate (Sinopharm Chemical Reagent Co., Ltd., 10019408), and lead nitrate (Sigma, 203580).

[0300] (2) Establishment of tumor model

[0301] The specific experimental steps are as follows:

[0302] Balb / c-nude female mice aged 6 to 8 weeks, with a specific pathogen-free grade and a body weight of 18±2g were selected and purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Certificate No.: [Lu] 2022-0006). All experimental procedures were strictly in accordance with the "Regulations on the Management of Experimental Animals" promulgated by the Experimental Animal Ethics Committee of Shandong First Medical University. All mice were provided with standard laboratory diet and water, a temperature of 25±2°C, a light / dark cycle of 12h, and adapted to the environment for at least one week. Female Balb / c-nude mice were randomly divided into 2 groups, with 10 mice in each group. The concentration of 1.0×10 7 The TOV-21G cell suspension was prepared with normal saline containing 100 cells / mL. The transplanted TOV-21G cells were inoculated subcutaneously into the scapula of mice (0.15 mL per mouse) to establish a tumor-bearing mouse model. After 24 hours of rest, the tumor size in the model group was 12.7±2.52 mm 3 (p<0.01, compared with the blank control group mice), the tumor-bearing mice were randomly divided into the model group (MG) and the ivermectin treatment group (TG).

[0303] The mice in the model group were gavaged with an equal amount of normal saline, and the mice in the ivermectin treatment group were gavaged with 10 mg / kg. Ivermectin was fully ground in a mortar and pestle in a sterile kitchen and then added to normal saline to make a suspension for administration. Each group of mice was treated once a day for 15 consecutive days. The changes in the body weight, tumor weight, and tumor volume of the test mice were statistically analyzed. After 15 days of continuous administration, the mice were killed, and the solid tumors of the mice were taken and minced into 2 mm pieces with a sterile scalpel. 3 After the pieces are sized, they are immediately embedded in electron microscopy fixative and set aside.

[0304] (3) TEM preparation steps

[0305] 3.1 Sampling and fixation: Determine the sampling site with fresh tissue, minimize mechanical damage such as pulling, contusion and squeezing, and sample within 2 minutes. Sample tissue should be 1mm 3 Before sampling, a culture dish filled with electron microscope fixative can be prepared in advance. After the small tissue block is removed from the body, it is immediately placed in the culture dish and cut into 1mm pieces in the fixative of the culture dish with a scalpel. 3 Then transfer the cut small tissue blocks to EP tubes filled with new electron microscope fixative for further fixation, and store and transport them at 4℃.

[0306] The plate was washed three times with 0.1 M phosphate buffer PB (pH 7.4), each time for 15 min.

[0307] 3.2 Post-fixation: Fix with 1% osmium hydroxide in 0.1 M phosphate buffer (PB) (pH 7.4) for 2 h at room temperature in the dark. Rinse three times with 0.1 M phosphate buffer (PB) (pH 7.4), 15 min each time.

[0308] 3.3 Dehydration at room temperature: Dehydrate the tissue in ascending steps of 30%-50%-70%-80%-95%-100%-100% alcohol for 20 minutes each, and 100% acetone twice for 15 minutes each.

[0309] 3.4 Infiltration and embedding: Acetone: 812 embedding medium = 1:1, 37°C, 2-4 hours, Acetone: 812 embedding medium = 1:2, 37°C, overnight, Pure 812 embedding medium at 37°C, 5-8 hours. Pour pure 812 embedding medium into the embedding plate, insert the sample into the embedding plate, and incubate in a 37°C oven overnight.

[0310] 3.5 Polymerization: Place the embedded plate in a 60℃ oven for polymerization for 48 hours, then remove the resin block for later use.

[0311] 3.6 Positioning: The resin block was sliced ​​into 1.5 μm semi-thin sections using a semi-thin microtome, stained with toluidine blue, and positioned under a light microscope.

[0312] 3.7 Ultrathin sectioning: The resin block was sliced ​​into 60-80 nm thin slices using an ultrathin microtome, and the slices were picked up using a 150-mesh copper mesh.

[0313] 3.8 Staining: Stain the copper grid in 2% uranyl acetate saturated alcohol solution in the dark for 8 minutes. Rinse three times with 70% alcohol and ultrapure water. Stain the grid in 2.6% lead citrate solution in the dark for 8 minutes. Rinse three times with ultrapure water and blot dry with filter paper. Place the copper grid sections in a grid box and dry overnight at room temperature.

[0314] 3.9 Observe under a transmission electron microscope and collect images for analysis.

[0315] Transmission electron microscopy observation of mitochondrial morphology in mouse ovarian cancer tissue Figure 27 As shown in the figure, A represents the electron microscopic observation pictures of ovarian cancer tissues with different treatments, and the magnifications from left to right are 2500X, 8000X and 20000X, respectively; B represents the quantitative analysis of mitochondrial autophagy, MG represents the normal saline control group, TG represents the ivermectin treatment group, *** represents P < 0.001 compared with the model group MG.

[0316] according to Figure 27It can be seen that after tumor-bearing mice were treated with ivermectin (10 mg / kg) for 15 consecutive days, the mitochondria in the tumor tissue of the TG group mice showed significant autophagy. Specifically, the mitochondria were wrapped by autophagosomes and surrounded by additional membrane structures. In terms of morphological changes, the mitochondria in the TG group showed swelling or deformation, and irregular shape. The inner and outer membranes were incomplete, the cristae were blurred or disappeared, the mitochondrial contents were degraded, and mitochondrial vacuolation and uneven contents could be clearly observed. In contrast, the mitochondria in the tumor tissue of mice that were not treated with ivermectin (given the same amount of normal saline as the control) were oval or rod-shaped, with a clear double-layer membrane structure, a smooth outer membrane, and an inner membrane that folded inward to form cristae. The internal matrix density was uniform, and some electron-dense particles could be seen. The analysis showed that ivermectin had a significant effect on the morphology and function of mitochondria in ovarian cancer cells TOV-21G. It can be clearly seen that the mitochondria in the MG group were oval or rod-shaped, with a clear double-layer membrane structure. The outer membrane is smooth, and the inner membrane folds inward to form cristae. The cristae increase the surface area of ​​the inner membrane and help with energy production. The density of the internal matrix is ​​uniform, and some electron-dense particles can be seen. These are enzyme complexes involved in oxidative phosphorylation. The mitochondria in the TG group are wrapped by autophagosomes and surrounded by additional membrane structures. In terms of morphological changes, the mitochondria in the TG group appeared swollen or deformed, with irregular shapes. The integrity of the inner and outer membranes was destroyed, and the cristae became blurred or disappeared. The contents of the mitochondria were degraded, manifested as changes in electron density, vacuolation, or uneven contents. The mitochondria in the TG group showed significant autophagy, in sharp contrast to the normal mitochondria in the MG group.

[0317] Example 5: Screening of Ivermectin Target Proteins Using Click Chemistry Technology

[0318] As can be seen from Examples 1 to 4, the present application establishes a protein change spectrum of ivermectin in vivo treatment of ovarian cancer, reveals the mechanism network of ivermectin's anti-ovarian cancer effect, and analyzes the data of ovarian cancer cells TOV-21G treated with ivermectin, revealing the intervention effect of ivermectin on the energy metabolism pathway of ovarian cancer. Based on the in vivo study of the anti-ovarian cancer effect of ivermectin in experimental animals, the mechanism of ivermectin's anti-ovarian cancer effect in humans is reflected.

[0319] Chemical proteomics is an emerging technology that uses small molecule compound modification and proteomic analysis to identify the protein targets of small molecule compounds in living cells. It has important research and application value in disease and drug research. Its principle is to transform small molecule compounds into labeled probes containing protein enrichment tags without changing the activity of small molecule compounds, and directly treat living cells to enrich proteins that interact with small molecule compounds in cells. Protein targets are identified through proteomic analysis. The screening test process is as follows: Figure 28The specific method is as follows:

[0320] 1. Molecular probe binding to ivermectin

[0321] Click chemistry technology was used to add molecular probes (composed of diazirine and alkynyl groups) to ivermectin, such as Figure 29 As shown in A, HPLC-MS spectrum and 1 H-NMR spectrum analysis, HPLC-MS spectrum analysis results are as follows Figure 29 As shown in B, 1 The results of H-NMR spectrum analysis are as follows Figure 30 As shown, A in the figure represents ivermectin 1 H-NMR spectrum, B represents ivermectin with molecular probe 1 H-NMR spectrum.

[0322] according to Figure 29 and Figure 30 It can be seen that the molecular probe was successfully combined with ivermectin.

[0323] 2. Cell Experiment Analysis

[0324] The experimental groups were: probe group, negative control group, and competition group. The probe group added a molecular probe composed of diazirine and alkynyl groups to target small molecule binding proteins. The negative control group did not add DMSO to remove background proteins such as biotin binding proteins. The competition group added ivermectin with a molecular probe. The competition group was treated at the same concentration as the probe group, while a large amount of the original molecule was added. The original molecule competed with the probe for binding to its specific binding protein. In this way, the differentially expressed proteins obtained by comparative analysis between the competition group and the probe group were the specific binding proteins of the small molecule.

[0325] The optimal protein binding concentration and the optimal competitive concentration of the probe were determined by in-gel fluorescence experiments. Then, three biological replicates were set up in each group, and a total of 9 samples were tested for protein spectrum. The samples were run on the separation gel until the separation gel was about 3-5 mm, and the electrophoresis was stopped. The gel was cut into 1 mm 3 The small pieces were placed in a 1.5 mL EP tube and decolorized, reduced, alkylated, and enzymatically digested overnight. The peptides were finally extracted and processed by mass spectrometry and bioinformatics analysis to screen for target binding proteins. The results of the in-gel fluorescence experiment are shown in Figure 2. Figure 31 As shown in the figure, A represents the intra-gel fluorescence image of the molecular probe at different concentrations, B represents the experimental results of different concentrations of the molecular probe, C represents the intra-gel fluorescence image of different probe-drug ratios in the competition group, D represents the screening experimental results of different competition concentration ratios in the competition group, and E represents the survival rate of cells treated with the molecular probe ivermectin.

[0326] Based on the Raw files obtained from mass spectrometry detection, the corresponding database is searched, and then protein identification is performed based on the results of the database search. At the same time, peptide, protein and parent ion mass tolerance distribution analysis is performed to evaluate the quality of mass spectrometry detection data; then protein quantitative analysis is performed, including overall difference analysis of identified proteins and screening of differential proteins and expression pattern clustering analysis; then the identified proteins are annotated with common functional databases, including COG / KOG, GO and KEGG databases; finally, a series of differential protein functional analyses such as GO and KEGG functional enrichment analysis and interaction network analysis are performed on the screened differential proteins.

[0327] Protein qualitative analysis showed that 319 proteins were targeted and fished out. According to the 2-fold difference, 32 differentially expressed proteins were identified, including SERPINA, IGKC, IGHG1, IGHA1, (FIBA)FGA, (FIBG)FGG, PDIA1, (IGLC3)IGL@, (XRCC6)G22P1, RAC1, (SYWCX)WARS1, (EFIB)EEF1B2, (PSA2)PSMA 2, (1433B)YWHAB, (1433S)SFN, (GLYM)SHMT2, (RL28)RPL28, (IMA1)KPNA2, (GDIR1)ARHG DIA, (PSA6)PSMA6, RAB10, PSME3, (RS14)RPS14, (RL23A)RPL23A, (TADBP)TARDBP, (PS MD2)TRAP2, (TIF1B)TRIM28, CNN2, (ZC3HD)ZC3H13, (SPB120)SERPINB12, (TRAP1)TGFBR AP1, HINT1.

[0328] 3. Validation Analysis

[0329] 3.1Survival Analysis

[0330] The 32 molecular proteins obtained by click chemistry screening were subjected to survival analysis. The analysis results are as follows: Figures 32-39 As shown, Figure 32 Survival analysis results of SERPINA, IGKC, IGHG1, and IGHA1 molecular proteins. Figure 33 Survival analysis results of FGA, FGG, PDIA1, and IGH@molecular proteins. Figure 34 Survival analysis results of G22P1, RAC1, WARS, and EEF1B2 molecular proteins. Figure 35Survival analysis results of PSMA2, YWHAB, SFN, and SHMT2 molecular proteins. Figure 36 Survival analysis results of RPL28, KPNA2, ARHGDIA, and PSMA6 molecular proteins. Figure 37 Survival analysis results of RAB10, PSME3, RPS14, and RPL23A molecular proteins. Figure 38 Survival analysis results of TARDBP, TRAP2, TRIM28, and CNN2 molecular proteins. Figure 39 The survival analysis results of ZC3H13, SERPINB12, TGFBRAP1, and HINT1 molecular proteins are shown in Table 6.

[0331] Table 632 Survival results of molecular proteins

[0332]

[0333]

[0334] according to Figures 32-39 As shown in Table 6, IGHA1, (FIBA)FGA, (FIBG)FGG, PDIA1, (IGLC3)IGL@, (SYWCX)WARS1, (EFIB)EEF1B2, (1433B)YWHAB, (1433S)SFN, (RL28)RPL28, (GDIR1)ARHGDIA, (PSA6)PSMA6, (RS14)RPS14, (RL23A)RPL23A, (TADBP)TARDBP, (PSMD2)TRAP2, (SPB120)SERPINB12, (TRAP1)TGFBRAP1 and HINT1 proteins were significantly correlated with the survival of ovarian cancer cells.

[0335] 3.2 Molecular docking experiment

[0336] Literature has confirmed that ivermectin interacts with ACE2, TMPRSS2, and MPro. Therefore, we used Discovery Studio Libdock software to analyze the interactions between ivermectin and ACE2, TMPRSS2, and MPro, and the molecular docking results are shown in Figure 2. Figures 40-42 As shown, Figure 40 This is the molecular docking diagram of ACE2 and ivermectin. Figure 41 This is the molecular docking diagram of TMPRSS2 and ivermectin. Figure 42 This is the molecular docking diagram of MPro and ivermectin.

[0337] The molecular docking results of ivermectin with ACE2, TMPRSS2 and MPro were used as positive controls. The 32 molecular proteins obtained by click chemistry screening were molecularly docked with ivermectin and protein molecules using Discovery Studio Libdock software. The results are shown in Table 7.

[0338] Table 7 Molecular docking results of 32 molecular proteins and ivermectin

[0339]

[0340]

[0341] According to Table 7, SERPINA, IGHG1, IGHA1, (FIBA) FGA, (FIBG) FGG, PDIA1, (IGLC3) IGL@, (XRCC6) G22P1, RAC1, (PSA2) PSMA2, (1433B) YWHAB, (1433S) SFN, (GLYM) SHMT2, (RL28) RPL28, (IMA1) KPNA2, (GDIR1) ARHGDIA, (PSA6) PSMA6, RAB10, PSME3, (RS14) RPS14, (RL23A) RPL23A, (TADBP) TARDBP, (PSMD2) TRAP2, CNN2, (ZC3HD) ZC3H13, (TRAP1) TGFBRAP1, and HINT1 can be molecularly docked with ivermectin. The molecular docking diagram is shown in FIG. Figures 43-69 shown. Figure 43 This is the molecular docking diagram of SERPINA and ivermectin. Figure 44 This is the molecular docking diagram of IGHG1 and ivermectin. Figure 45 This is the molecular docking diagram of IGHA1 and ivermectin. Figure 46 This is the molecular docking diagram of FGA and ivermectin. Figure 47 This is the molecular docking diagram of FGG and ivermectin. Figure 48 This is the molecular docking diagram of PDIA1 and ivermectin. Figure 49 This is the molecular docking diagram of (IGLC3)IGL@ and ivermectin. Figure 50 This is the molecular docking diagram of (XRCC6)G22P1 and ivermectin. Figure 51 This is the molecular docking diagram of RAC1 and ivermectin. Figure 52 This is the molecular docking diagram of (PSA2)PSMA2 and ivermectin, Figure 53 This is the molecular docking diagram of (1433B)YWHAB and ivermectin. Figure 54 This is the molecular docking diagram of (1433S)SFN and ivermectin. Figure 55This is the molecular docking diagram of (GLYM)SHMT2 and ivermectin. Figure 56 This is the molecular docking diagram of (RL28)RPL28 and ivermectin, Figure 57 This is the molecular docking diagram of (IMA1)KPNA2 and ivermectin. Figure 58 This is the molecular docking diagram of (GDIR1)ARHGDIA and ivermectin. Figure 59 Molecular docking diagram of (PSA6)PSMA6 and ivermectin, Figure 60 This is the molecular docking diagram of RAB10 and ivermectin. Figure 61 This is the molecular docking diagram of PSME3 and ivermectin. Figure 62 This is the molecular docking diagram of (RS14)RPS14 and ivermectin. Figure 63 This is the molecular docking diagram of (RL23A) RPL23A and ivermectin, Figure 64 This is the molecular docking diagram of (TADBP)TARDBP and ivermectin. Figure 65 This is the molecular docking diagram of (PSMD2)TRAP2 and ivermectin, Figure 66 This is the molecular docking diagram of CNN2 and ivermectin. Figure 67 This is the molecular docking diagram of (ZC3HD)ZC3H13 and ivermectin. Figure 68 This is the molecular docking diagram of (TRAP1)TGFBRAP1 and ivermectin. Figure 69 This is the molecular docking diagram of HINT1 and ivermectin.

[0342] In summary, SERPINA, IGHG1, IGHA1, (FIBA)FGA, (FIBG)FGG, PDIA1, (IGLC3)IGL@, (XRCC6)G22P1, RAC1, (PSA2)PSMA2, (1433B)YWHAB, (1433S)SFN, (GLYM)SHMT2, (RL28)RPL28, (IMA1)KPNA2, (GDIR1)ARHGDIA, (PSA6)PSMA6, RAB10, PSME3, (RS14)RPS14, (RL23A)RPL23A, (TADBP)TARDBP, (PSMD2)TRAP2, CNN2, (ZC3HD)ZC3H13, (TRAP1)TGFBRAP1, and HINT1 can be used as target proteins of ivermectin for ovarian cancer cells.

[0343] As can be seen from the above examples, the present invention provides an ivermectin target protein and its application. The present invention establishes the protein change spectrum of ivermectin in vivo treatment of ovarian cancer, reveals the mechanistic network of ivermectin's anti-ovarian cancer effect, and analyzes the data of ivermectin-treated ovarian cancer cell TOV-21G to reveal the intervention effect of ivermectin on the energy metabolism pathway of ovarian cancer. The present invention evaluates the anti-ovarian cancer effect of ivermectin in vitro and in vivo and its regulatory effect on energy metabolism. Through "click chemistry" technology, fluorescent probe technology and target fishing, the target protein of ivermectin on ovarian cancer cells is determined. Then, further analysis is conducted to obtain the ivermectin target protein, which can be used for the prediction, preclinical prevention and personalized medication of ovarian cancer, and promote the development of clinical medication for ovarian cancer under the medical framework.

[0344] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A drug target protein for ovarian cancer, characterized in that: The drug target proteins include one or more of SERPINA, IGHG1, IGHA1, FGA, FGG, PDIA1, IGLC3, G22P1, RAC1, PSMA2, YWHAB, SFN, SHMT2, RPL28, IMA1, ARHGDIA, PSMA6, RAB10, PSME3, RPS14, RPL23A, TARDBP, TRAP2, CNN2, ZC3H13, TGFBRAP1 and HINT1.

2. Use of the drug target protein according to claim 1 as a disease marker for ovarian cancer.

3. Use of a reagent for detecting the drug target protein according to claim 1 in preparing a kit for diagnosing ovarian cancer.

4. Use of a reagent for detecting the drug target protein according to claim 1 in preparing a kit for evaluating the prognosis of ovarian cancer.

5. Use of the drug target protein according to claim 1 as a screening target in screening drugs for treating ovarian cancer.

6. Use of ivermectin in the preparation of a medicament for treating ovarian cancer.

7. The use according to claim 6, characterized in that The dosage of ivermectin is 8-12 mg / kg.

8. The use according to claim 6, characterized in that The ivermectin regulates the energy metabolism of ovarian cancer cells by acting on the drug target protein, thereby inhibiting the proliferation of ovarian cancer cells.

9. The use according to claim 8, characterized in that The drug target protein is the drug target protein according to claim 1.

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