Application of sophoricoside in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses application of sophoricoside in preparation of a medicine for treating hepatocellular carcinoma. The influence of sophoricoside single medicine or combination of sophoricoside and lenvatinib on HCC cells is analyzed through in-vitro and in-vivo experiments (clone formation, CCK-8, flow cytometry and a xenograft model). A potential target spot is predicted through a protein interaction network (PPI) and KEGG enrichment, and verification is carried out through molecular docking, surface plasma resonance (SPR), ubiquitination and Western blot. Results show that Sop treatment can inhibit survival and proliferation of HCC cells and induce cell cycle arrest (in vitro and in vivo). The sophoricoside and the lenvatinib are combined for use, so that a synergistic anti-HCC effect is achieved.

Description

Technical Field

[0001] The invention relates to application of sophora japonica glycoside in preparing medicine for treating hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. More than 60% of HCC patients have progressed to the middle and late stages when diagnosed, and have lost the opportunity for radical surgery. Systemic treatment has become its main treatment option. In this context, traditional Chinese medicine treatment has gradually become an important part of the comprehensive treatment system of HCC with its advantages of multi-target and overall regulation. Among them, Chinese medicine monomers provide ideal research objects for modern Chinese medicine research because of their clear chemical composition and explorable mechanism of action. Sophora japonica glycoside (SOP) is an isoflavone glycoside compound isolated from traditional Chinese medicines such as Sophora japonica flowers and Sophora japonica horns. Previous studies have shown that the compound exhibits significant anti-inflammatory and antioxidant activities, but its potential value still needs to be further explored. Summary of the invention

[0003] The main purpose of the present invention is to provide the use of sophora japonica glycoside in the preparation of drugs for treating hepatocellular carcinoma.

[0004] Furthermore, the drug also includes lenvatinib.

[0005] Furthermore, sophora japonica glycosides inhibited HCC cell survival and proliferation and induced cell cycle arrest.

[0006] Another object of the present invention is to provide a drug for treating hepatocellular carcinoma, wherein the drug comprises an effective therapeutic amount of sophoroside.

[0007] Furthermore, the drug also includes lenvatinib.

[0008] Furthermore, the sophora japonica glycosides and lenvatinib in the drug significantly synergistically treat hepatocellular carcinoma.

[0009] Another object of the present invention is to provide the use of sophora japonica glycosides in the preparation of a hepatocyte cycle arrest inducer or a hepatocyte EGFR inhibitor.

[0010] In this invention, in vitro and in vivo experiments (colony formation, CCK-8, flow cytometry and xenograft models) were conducted to analyze the effects of sophocarpine alone or in combination with lenvatinib on HCC cells. Potential targets were predicted through protein-protein interaction network (PPI) and KEGG enrichment, and verified through molecular docking, surface plasmon resonance (SPR), ubiquitination and Western blot. The experimental results demonstrated that sophocarpine treatment could inhibit the survival and proliferation of HCC cells and induce cell cycle arrest (in vitro and in vivo). The combination of sophocarpine and lenvatinib had a synergistic anti-HCC effect. Sophocarpine directly bound to EGFR and induced its degradation, enhancing the efficacy of lenvatinib by inhibiting the EGFR-AKT / STAT3 signaling pathway. Description of the Drawings

[0011] The present invention will be further described below in conjunction with the drawings and embodiments.

[0012] Figure 1 Effect of sophocarpine on the proliferation of Huh7 and SK-Hep-1

[0013] A. The half-inhibitory concentration of sophocarpine in Huh7 and SK-Hep-1 was determined by CCK8 assay. B. The inhibitory effect of sophocarpine on the growth of Huh7 and SK-Hep-1 was measured by CCK8 assay at sophocarpine concentrations of 0, 300, and 600 μM. C. The effect of sophocarpine on the proliferation of Huh7 and SK-Hep-1 under the intervention of the above concentrations was determined by colony formation assay, and the colony formation was counted.

[0014] Figure 2 Sophocarpine induces cell cycle arrest in Huh7 and SK-Hep-1 cells

[0015] A. After treating Huh7 and SK-Hep-1 cells with sophocarpine for 48 hours, flow cytometry analysis was performed to count the cell cycle. B. The expression of PCNA, CDK1 and Cyclin B1 was further detected by Western blot.

[0016] Figure 3 Sophocarpine inhibits the growth of tumors in nude mice

[0017] A. Photos of Huh7 xenograft tumors under sophocarpine treatment; B. Tumor growth curves under sophocarpine treatment; C. Final tumor weights after sophocarpine treatment. D. Detection of the contents of ki67 and CDK1 in tumor tissues of nude mouse Huh7 xenografts by immunohistochemistry, scale bar: 100 μm (left) and 50 μm (right). E. Analysis of the expression levels of the proteins in xenograft tumors by Western blotting. F. Animal body weight gain curves under sophocarpine treatment. G. HE staining of the organs of nude mice in the drug administration group and the control group (scale bar: 100 μm). *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 4 Combined drugs synergistically inhibit the proliferation of HCC cells

[0019] A. and B. The IC50 of lenvatinib alone or in combination with 75 μM or 150 μM sophocarpine was evaluated in Huh7 and SK-Hep-1 cells by CCK-8 assay. The CI values of combined administration at different doses were calculated using CompuSyn software. C. The CCK-8 assay was used to determine the effects of lenvatinib, sophocarpine, and combination therapy on the proliferation of Huh7 and SK-Hep-1 cells. The drug administration concentrations of the two cell lines in the CCK-8 assay were as follows: lenvatinib (Huh7: 1 μM, SK-Hep-1: 6 μM), sophocarpine (Huh7: 100 μM, SK-Hep-1: 100 μM). D. The effects of lenvatinib, sophocarpine, and combined drugs on the proliferation of Huh7 and SK-Hep-1 cells were detected by colony formation assay, and the number of colonies was counted. The drug administration concentrations of the two cell lines in the colony formation assay were as follows: lenvatinib (Huh7: 1 μM, SK-Hep-1: 6 μM), sophocarpine (Huh7: 100 μM, SK-Hep-1: 100 μM).

[0020] Figure 5 Combined drugs downregulate the EGFR-AKT / STAT3 pathway and induce apoptosis of HCC cells

[0021] A. Treat Huh7 and SK-Hep-1 cells with lenvatinib (Huh7: 1 μM, SK-Hep-1: 6 μM), sophoricoside (Huh7: 100 μM, SK-Hep-1: 100 μM) and combination treatment for 48 hours. Then, flow cytometry analysis was performed to monitor apoptosis, and statistical analysis was carried out on the number of apoptotic cells in each group. B. Treat Huh7 and SK-Hep-1 cells with different drugs. Western-blot was performed to monitor the expression of Cleaved-caspase-3 and Cleaved-PARP1. C. Treat Huh7 and SK-Hep-1 cells with different drugs. Western-blot was used to detect the expression of EGFR, pEGFR, AKT, pAKT, STAT3, pSTAT3. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 6 In vivo anti-HCC effect of combination treatment

[0023] A. Tumor photos of Huh7 xenografts under different treatment methods, B. Final tumor weights after different treatments. C. Tumor volume growth curves under different treatment methods. D. Body weight growth curves of nude mice under different treatment methods. E. Western blot analysis was performed to detect the expression of EGFR and downstream pathway proteins AKT and STAT3 in xenograft tumors; protein quantification in each group. F. Western blot analysis was performed to detect the expression and quantitative analysis of apoptotic proteins in xenograft tumors. (*P<0.05, **P<0.01, ***P<0.001, ns: no statistical significance). Detailed implementation manners

[0024] Example 1

[0025] Experimental materials Sophoricoside: The purified reagent purchased from TargetMol, with a purity of over 99%.

[0026] 1. Cell culture

[0027] Both SK-Hep-1 and Huh-7 cells used in the present invention are adherent HCC cells, and are cultured in adherent culture dishes. The cell culture medium is a mixture of 10% fetal bovine serum, 1% penicillin-streptomycin mixture and DMEM medium, and is cultured at 37 °C and 5% CO2. And cell treatment-related operations are completed in a biosafety cabinet.

[0028] After cell resuscitation, add 1 mL of complete medium to the centrifuge tube, blow it evenly, and then evenly drop the cell suspension into the culture dish. Add 6 - 8 mL of complete medium to the culture dish, slowly shake the culture dish in the front-back, left-right directions to mix the cells evenly, and then put it into the incubator for culture.

[0029] 2 Clonogenic assay

[0030] (1) When the cell state is normal and the cells grow to 70 - 80%, perform trypsin digestion. After using 2 mL of complete medium to terminate the digestion, transfer the cell suspension to a 10 mL centrifuge tube.

[0031] (2) Subsequently, take an appropriate amount of the cell suspension for the following experiments. Centrifuge a 5 mL EP tube at 1000 rpm for 3 minutes, remove the supernatant, add 1 mL of PBS for washing, mix well, centrifuge again under the same conditions, and discard the supernatant.

[0032] (3) Then add 1 mL of basal medium to resuspend the cells. Take an appropriate amount of the cell suspension for counting, seed the cells into a six - well plate at a density of 2500 cells per well, and set up the experimental system: blank control group (DMSO), sophoricoside monotherapy group, lenvatinib monotherapy group, and combination drug administration group; set 3 technical replicates for each group, mix well and place in an incubator for 11 - 14 days of culture.

[0033] (4) When more than 50 colonies are formed, add 1 mL of 4% paraformaldehyde solution and fix at room temperature for 30 minutes, then remove the fixative.

[0034] (5) Slowly add crystal violet stain along the side wall of the culture dish and stain for 20 minutes.

[0035] (6) After staining, gently rinse from the side wall with PBS buffer, then slowly rinse under tap water and dry in an oven. The clone morphology will be clearly visible, and then perform image acquisition, quantity statistics, and result illustration.

[0036] 3 CCK8 and cell number detection

[0037] (1) After digestion, centrifuge the cells at 1000 rpm for 3 min, discard the supernatant, add 1 mL of complete medium and mix well.

[0038] (2) Prepare a 1.5 mL centrifuge tube. Mix 10 μL of trypan blue and 10 μL of the cell suspension, then aspirate 10 μL of the mixture and drop it on one side of the cell counting chamber.

[0039] (3) Seed 4000 Huh - 7 cells and SK - Hep - 1 cells per well. Aspirate a quantitative cell suspension according to the cell number, add complete medium according to the required number of wells, and seed into a 96 - well plate for culture, with a volume of 100 μL per well.

[0040] (4) After the cells adhere, change to the medium containing the experimental drug for culture, and set the co - culture duration of the cells according to the drug time - effect relationship.

[0041] (5) At fixed intervals of time, 100 μL of basal culture medium and 10 μL of CCK-8 reagent were injected into each well.

[0042] (6) The samples were incubated in an incubator for 90 minutes, and then the OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The experimental data were plotted using GraphPad prism9 software.

[0043] 4 Detection of cell apoptosis

[0044] (1) The supernatant in the culture dish or plate was aspirated and transferred to a 10 mL centrifuge tube.

[0045] (2) 1 mL of PBS was added along the culture dish to wash the cells twice, and the PBS was collected in the centrifuge tube.

[0046] (3) 1 mL of trypsin without EDTA was added to digest the cells. Observation was carried out under a microscope until the cell gaps disappeared and most of the cells detached. The trypsin was collected in the centrifuge tube.

[0047] (4) The culture dish was tapped to observe that all the cells had detached. 2 mL of culture medium was added to transfer the cell suspension to the centrifuge tube. The cells were centrifuged at 2000 rpm for 3 min, and the supernatant was discarded.

[0048] (5) 2 mL of PBS was added and the cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. This step was repeated once.

[0049] (6) 1×Annexin V Binding Solution prepared in advance according to the instructions was added; 5 μL of Annexin V, FITC conjugate was added to the cell suspension, and then 5 μL of PI Solution was added. The cells were cultured in the dark at room temperature for 15 min.

[0050] (7) Finally, 400 μL of 1×Annexin V Binding Solution was added, and the cells were detected using a flow cytometer (Beckman) within 1 h. The data were analyzed using Kaluza software.

[0051] 5 Detection of cell cycle

[0052] (1) Cells in good condition were seeded into a 6 cm culture dish and cultured. After the cells adhered to the dish, experimental operations were carried out.

[0053] (2) After the experiment, the culture was stopped. After digesting the cells, 1 mL of the suspension was taken and placed into a 1.5 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 3 minutes.

[0054] (3) Discard the supernatant, and add 1 mL of 70% ethanol stored at -20°C to the cell pellet. Vortex to disperse the cells and let stand at 4°C for 2 h;

[0055] (4) Take out the sample at 4°C, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, wash the cells with 1 mL of PBS and then centrifuge again for 3 minutes;

[0056] (5) After discarding the supernatant, add the working solution prepared according to the instructions, vortex to disperse the cells, and incubate in the dark at 37°C in a constant temperature oven for 30 min;

[0057] (6) Take out the sample from the oven and vortex, add the dye and incubate in the dark at 4°C for 30 min, vortex again to disperse the cells, and filter through a nylon mesh to remove cell clumps in the sample; Detect by flow cytometry.

[0058] 6 Protein-related experiments

[0059] Total cell protein extraction:

[0060] (1) Protein extraction should be carried out on ice throughout the process. Prepare the protein lysis buffer containing protease inhibitors in advance and pre-cool it on ice. Add protease inhibitors in a ratio of 100:1. If phosphorylated proteins are to be extracted, add phosphatase inhibitors in the same ratio;

[0061] (2) After cell treatment, aspirate the culture medium, wash twice with PBS, place on ice, add an appropriate amount of protein lysis buffer, shake well and place on ice for ten minutes; At this time, pre-cool the centrifuge to 4°C in advance;

[0062] (3) Try to collect the lysed protein into a 1.5 mL EP tube, turn on the ultrasonic crusher, adjust the power to 20%, and set the time interval to 5 seconds. Perform ultrasonic treatment twice according to the power and time, and then place on ice for 10 minutes.

[0063] (4) Use a vortex shaker to shake the cell suspension for 5 seconds, with 3 shakes, and a 10-minute interval between each shake;

[0064] (5) After shaking, centrifuge at 4°C and 12000 rpm for 10 min using a low-temperature centrifuge, and slowly aspirate the upper layer of liquid into a new EP tube. The upper layer of liquid at this time is the extracted protein;

[0065] (6) Determine the protein concentration by the BCA method. Prepare the BCA working solution according to the kit instructions. Pipette 200 μL of the BCA working solution into each well of a 96-well plate, and then pipette 10 μL of the above protein supernatant into the well. Incubate in a 37°C oven for 30 minutes;

[0066] (7) Set the parameters of the microplate reader, measure the absorbance of each well at 562 nm, and use the protein standard curve to determine the concentration of the protein sample;

[0067] (8) Add 5×loading buffer and lysis buffer to the protein supernatant to balance the protein concentration, then place it in a 100°C metal bath and boil the protein for 10 minutes. The boiled protein supernatant can be stored at 4°C for one week or at -20°C for several weeks, but try to avoid repeated freezing and thawing that may cause protein degradation.

[0068] SDS-PAGE electrophoresis: Conduct it according to the conventional method.

[0069] 7 Protein stability test

[0070] (1) After the cell experiment treatment is completed, add cycloheximide (CHX) to the complete medium at a concentration of 200 μg / mL, and then set the intervention times of 0, 4, 8, and 12 hours; after completion, aspirate the medium and wash twice with PBS.

[0071] (2) Extract the total cellular protein and perform SDS-PAGE electrophoresis according to the above experimental method.

[0072] 8 Pathological staining

[0073] Immunohistochemical staining: Conduct it according to the conventional method.

[0074] HE staining: Conduct it according to the conventional method.

[0075] SPR molecular affinity test

[0076] (1) Export the two-dimensional structure of sophoricoside from the PubChem database, and at the same time convert it into a three-dimensional format using Chemdraw software. Export the crystal structure of the core target from the PDB database, and then use the iGEMDOCK tool to dock the target protein with sophoricoside;

[0077] (2) Install the COOH chip according to the standard operating procedure of the OpenSPRTM instrument. Use PBST as the detection buffer and start running at a maximum flow rate of 150 μL / min. After reaching the signal baseline, sample 200 μL of IPA (isopropyl alcohol), run for 10 seconds to expel air bubbles, wash the sample loop with buffer after reaching the baseline, and empty it with air.

[0078] (3) After reaching the signal baseline, set the buffer flow rate to 20 μL / min and activate the chip with EDC / NHS solution (1:1). Incubate 200 μL of the sample in the immobilization solution for 4 minutes, rinse the sample loop with the buffer and empty the air. Add 200 μL of the blocking solution, rinse the sample loop with the buffer and empty the air. Dilute the analyte at the concentration shown in the experimental results with the analysis buffer and inject the sample at a rate of 20 μL / min. The analyte binds to the ligand for 240 seconds, and the ligand dissociates naturally for 360 seconds. Use TraceDrawer software for data analysis.

[0079] Protein ubiquitination experiment

[0080] (1) After treating HCC cells in the logarithmic growth phase with sophocarpine for 48 hours, add 10 μM proteasome inhibitor MG132 and continue to treat for 10 hours.

[0081] (2) Collect the cells, extract the proteins using IP lysis buffer containing protease inhibitors, bind the EGFR antibody to the immunomagnetic beads to form an antibody-immunomagnetic bead complex. Then add the antibody-immunomagnetic bead complex to the cell extract and incubate overnight on a shaker.

[0082] (3) Wash the antibody-immunomagnetic bead complex with the wash buffer multiple times to remove non-specifically bound proteins.

[0083] (4) Elute the protein, add 50 μL of 1× loading buffer (prepare the 5× loading buffer into 1× with WB / IP lysis buffer without protease inhibitors), and boil for 10 min in a metal bath at 100 °C.

[0084] (5) Separate the proteins by SDS-PAGE; use Western blotting to transfer the separated proteins onto a membrane for detection.

[0085] Animal experiment

[0086] Nude mouse subcutaneous tumor experiment

[0087] (1) Nude mice aged 3 - 4 weeks (purchased from Shanghai Slack) are first raised in an SPF environment for one week; all subsequent experiments are carried out in an SPF environment to avoid stress reactions caused by environmental changes.

[0088] (2) Cell culture: Seed the well-conditioned Huh7 cells into a 10 cm culture dish for amplification and passage to the target number. Maintain the activity and good growth conditions of the tumor cells (cell state directly affects the tumor formation effect), and change the fresh medium 24 hours before collection.

[0089] (3) Cell digestion: First, wash the cells with PBS, then digest them with trypsin and count. Wash again with PBS to completely remove serum residues. Finally, resuspend the cells in the basal medium, and take 3×10 6 cells and inoculate them subcutaneously in each nude mouse. The cells need to be placed on ice and inoculated within 30 - 60 minutes to ensure viability.

[0090] (4) Cell inoculation: Insert the needle 0.5 cm above the right axilla of the nude mouse and advance it about 0.5 cm forward to ensure no liquid leakage. Then, slowly inject 100 μL of the cell suspension subcutaneously, and gently pull out the needle after injection.

[0091] (5) Observation and grouping: Monitor tumor growth daily. After about 7 days, a subcutaneous mass will bulge. Divide the mice evenly according to tumor volume. In the experiment with sole use of sophoricoside, the nude mice are divided into a control group and a drug - administered group, with 4 mice in each group. In the experiment with combined treatment with lenvatinib, the nude mice are divided into four groups: a control group, a group treated with sole sophoricoside, a group treated with sole lenvatinib, and a group treated with combined sophoricoside and lenvatinib, with 4 mice in each group.

[0092] (6) Drug preparation and administration: In the experiment with sole use of sophoricoside, treat with sophoricoside at 40 mg / kg. In the combined - drug experiment, administer sophoricoside at 20 mg / kg and lenvatinib at 10 mg / kg. Administer the drugs daily during the experiment for a treatment course of 1 - 2 weeks. The two groups of mice are treated with different drugs, and the body weight and tumor volume changes are monitored regularly.

[0093] (7) After sacrificing the mice by cervical dislocation, collect the subcutaneous tumor tissues and take pictures. Divide the subcutaneous tumor tissues into two parts. The tissues used for extracting tissue proteome are stored at - 80 °C, and the tissues used for pathological tests are temporarily stored in formalin for subsequent experiments.

[0094] Extraction of subcutaneous tumor tissue protein

[0095] (1) Pre - cool the tissue grinding equipment;

[0096] (2) Weigh the tumor tissue, cut it into pieces and place it in an EP tube;

[0097] (3) Add an appropriate amount of RIPA lysis buffer to the EP tube (1000 μL for 0.05 g of tissue), then add 2 grinding beads with a diameter of 4 mm and perform 3 grinding treatments for 60 seconds each.

[0098] (4) Use an ultrasonic crusher to lyse the cells at 30% power

[0099] (5) Place the EP tube pre - cooled to 4 °C in a centrifuge and centrifuge at 12000 rpm for 10 - 20 minutes. Then quickly transfer the supernatant to a new EP tube and recover the grinding beads.

[0100] (6) After determining the protein solution concentration, add 5 times the loading buffer and heat in a metal bath at 100 °C for 10 minutes, and then use it for western-blot.

[0101] Statistical analysis of experimental results

[0102] All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Unless otherwise specified, data are expressed as mean ± standard deviation (SD). The differences between two groups were analyzed by t-test, and one-way ANOVA or Kruskal-Wallis test was used for comparisons among multiple groups. All legends were made using GraphPad Prism 9, Adobe Photoshop 2023, and Image J. (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: no statistical significance).

[0103] Experimental results

[0104] 1 Therapeutic effect of sophoricoside on HCC

[0105] 1.1 Sophoricoside inhibits HCC cell proliferation by inducing G2 / M phase arrest

[0106] To evaluate the inhibitory effect of sophoricoside on HCC proliferation, this application selected two of the most commonly used HCC cells, Huh7 and SK-Hep-1, for experiments. By detecting with the CCK-8 method combined with gradient concentration administration (0 - 800 μM, 48 h), it was found that sophoricoside could significantly inhibit the viability of HCC cells and showed a dose-dependent effect ( Figure 1 A). Through non-linear regression analysis, the half-maximal inhibitory concentration (IC50) of Huh7 and SK-Hep-1 cells was 358.46 ± 21.2 μM and 317.1 ± 24.7 μM, respectively. Based on this, three concentration groups of 0, 300, and 600 μM were further set up to verify the continuous inhibitory effect of sophoricoside through CCK-8 dynamic proliferation monitoring and colony formation experiments. The results showed that after 4 days of treatment with 600 μM sophoricoside, the survival rates of Huh7 and SK-Hep-1 cells decreased to 38.7% ± 6.7% and 43.68% ± 4.16% of the control group, respectively ( Figure 1 B), and after 14 days of sophoricoside intervention, the colony formation ability of the two cell lines was significantly inhibited ( Figure 1 C). The above results all indicate that sophoricoside has an inhibitory effect on the proliferation of HCC cells.

[0107] Based on the above-mentioned proliferation inhibitory phenotype, the specific mechanism by which sophocarpine regulates the cell cycle progression of HCC cells was further analyzed. First, the effect of sophocarpine on the growth cycle of HCC cells was detected. Through propidium iodide (PI) staining combined with flow cytometry analysis, it was found that treatment with sophocarpine for 48 h could significantly induce G2 / M phase arrest in Huh7 and SK-Hep-1 cells ( Figure 2 A); then, to further verify this result, Western blot detection was also performed on the proliferation marker PCNA and the G2 phase markers Cyclin B1 and CDK1. The results consistently showed that sophocarpine had a dose-dependent inhibitory effect on these proteins ( Figure 2 B). These findings together indicate that sophocarpine blocks the cell cycle progression of HCC cells and inhibits their proliferation by targeting the G2 / M phase checkpoint regulatory network.

[0108] 1.2 Sophocarpine inhibits the growth of subcutaneous tumors in nude mice

[0109] To further confirm the anti-cancer effect of sophocarpine in vivo, the applicant established a subcutaneous tumor model in nude mice and administered sophocarpine by gavage or an equal volume of vehicle control for 13 days. Analysis at the end of the experiment showed that the volume of subcutaneous tumors in the sophocarpine treatment group was significantly smaller than that in the control group ( Figure 3 A and B), and the average tumor weight was reduced by 60.6% ( Figure 3 C). In addition, immunohistochemical staining was used to detect the expression of the proliferation and cyclin Ki-67 and CDK1 in subcutaneous tumor tissues. The staining results showed that the levels of Ki-67 and CDK1 after treatment with sophocarpine were significantly decreased ( Figure 3 D), demonstrating that sophocarpine inhibited the proliferation of HCC cells in vivo and induced cell cycle arrest after administration. Western-blot detection of proliferation and cell cycle marker proteins also showed similar results ( Figure 3 E). In addition, the body weight of mice after treatment with sophocarpine did not decrease significantly compared with the control group ( Figure 3 F). At the same time, the HE staining results indicated that there were no obvious histological changes in the organs of nude mice after administration, suggesting that sophocarpine did not show toxic effects after administration in nude mice, indicating its good biosafety ( Figure 3 G).

[0110] 2 Therapeutic effect of the combination of sophocarpine and lenvatinib on HCC

[0111] 2.1 The combination of drugs synergistically inhibits the proliferation of HCC cells

[0112] Based on the key role of compensatory activation of EGFR signaling in lenvatinib resistance in liver cancer, the present invention proposes a scientific hypothesis that sophoricoside may enhance the sensitivity of targeted drugs by targeting and regulating EGFR. To verify this theory, the applicant designed a gradient concentration combination strategy to evaluate the synergistic effect of sophoricoside and lenvatinib in Huh7 and SK-Hep-1 cell lines. Drug sensitivity analysis showed that as the concentration of sophoricoside increased, the half-maximal inhibitory concentration (IC50) of lenvatinib decreased significantly in a dose-dependent manner ( Figure 4 A). In addition, Chou-Talalay combination index analysis using CompuSyn software found that the combination index (CI) of each concentration combination was less than 1 ( Figure 4 B), confirming a significant synergistic effect between the two drugs. Cell function experiments further corroborated this: CCK-8 assays showed that the cell survival rate in the combination group decreased compared to the single-drug groups ( Figure 4 C); colony formation assays indicated that combined intervention reduced the cell colony formation ability ( Figure 4 D), which was significantly better than the effect of single-drug treatment.

[0113] 2.2 Combined drugs downregulate the EGFR-AKT / STAT3 pathway and induce apoptosis in HCC cells

[0114] To systematically evaluate the synergistic antitumor effect of sophoricoside and lenvatinib, the effects of the two drugs alone and in combination on apoptosis of liver cancer cells were first detected by flow cytometry. The experimental data showed that although single treatment with sophoricoside had no significant effect on the apoptosis rate of Huh7 and SK-Hep-1 cells (P>0.05), compared with the single-lenvatinib group, combined treatment significantly increased the apoptosis rate (Huh7: 18.41% vs 10.22%, P<0.01; SK-Hep-1: 12.67% vs 6.07%, P<0.05) ( Figure 5 A). Consistent with this phenotype, Western blot analysis showed that the expression levels of the pro-apoptotic marker proteins cleaved PARP and cleaved caspase3 were significantly upregulated in the combined drug treatment group ( Figure 5 B). These results indicate that sophoricoside can effectively enhance the apoptosis-inducing effect of lenvatinib. This synergistic effect may be due to the blockade of compensatory activation of EGFR signaling by sophoricoside, thereby relieving the apoptosis resistance mechanism of HCC cells to lenvatinib.

[0115] The AKT and STAT3 pathways are downstream pathways of EGFR, and their overactivation is closely related to lenvatinib resistance. Therefore, their expression in HCC cells treated with sophoricoside, lenvatinib, and the combination of the two drugs was detected. Compared with the use of sophoricoside or lenvatinib alone, combined treatment with sophoricoside and lenvatinib significantly reduced the expression levels of pAKT and pSTAT3Figure 5 C). The above results indicate that sophoricoside can synergistically enhance the anti-cancer efficacy of lenvatinib by inhibiting EGFR-AKT and EGFR-STAT3 signal transduction.

[0116] 2.3 In vivo anti-HCC effect of combined drug use

[0117] To further evaluate the in vivo anti-HCC activity of the combination therapy of sophoricoside and lenvatinib, the applicant established a subcutaneous HCC xenograft model in nude mice. The mice were randomly divided into four groups: control group, sophoricoside monotherapy group, lenvatinib monotherapy group, and combination therapy group. After administration of sophoricoside and lenvatinib, the volume of subcutaneous tumors was measured every two days. The mice were sacrificed after 8 days of treatment, and the subcutaneous tumors of each group of mice were isolated and photographed ( Figure 6 A). Compared with the control group, the tumor volume and weight in the single drug treatment groups were significantly reduced. In addition, compared with the single drug groups, the tumor volume and weight in the combination drug treatment group were significantly reduced ( Figure 6 B and D). There was no significant difference in body weight among the groups, indicating good tolerance to the combined drug use ( Figure 6 C). Consistent with the in vitro research results, the Western-blot detection results of xenograft tumors showed that the combined use of sophoricoside and lenvatinib significantly down-regulated the expression of pAKT and pSTAT3 ( Figure 6 E), and at the same time increased the expression levels of Cleaved PARP and Cleaved Caspase3 ( Figure 6 F). These data indicate that sophoricoside can enhance the in vivo anti-HCC activity of lenvatinib.

[0118] As described above, it is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. Application of sophoricoside in the preparation of a drug for treating hepatocellular carcinoma.

2. The application according to claim 1, wherein The drug further comprises lenvatinib.

3. The application according to claim 1 or 2, characterized in that: Sophoricoside inhibits the survival and proliferation of HCC cells and induces cell cycle arrest.

4. A drug for treating hepatocellular carcinoma, characterized in that: The drug comprises an effective therapeutic amount of sophoricoside.

5. A drug for treating hepatocellular carcinoma according to claim 4, characterized in that: The drug further comprises lenvatinib.

6. The medicament for treating hepatocellular carcinoma according to claim 5, wherein: Sophoricoside and lenvatinib in the drug significantly synergistically treat hepatocellular carcinoma.

7. Application of sophoricoside in the preparation of a hepatocyte cycle arrest inducer or a hepatocyte EGFR inhibitor.

Images