Application of bergamottin in preparation of medicine for treating neuroblastoma

Abstract

The invention relates to the technical field of medical application, in particular to application of bergamottin in preparation of a medicine for treating neuroblastoma. The invention discloses the application of bergamottin in treatment of neuroblastoma for the first time, the invention further discloses the application of bergamottin targeting PLOD2 to play an anti-tumor role for the first time, and pharmacodynamic experiment data shows that the bergamottin BGM inhibits tumor growth and neuroblastoma metastasis by targeting PLOD2 expression, and can be used for treating neuroblastoma. Therefore, the bergamotin can be used as a targeted anti-NB potential drug, so that a new target spot is provided for the treatment of neuroblastoma.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical uses, and is an application of bergapten in the preparation of a drug for treating neuroblastoma. Background Art

[0002] Neuroblastoma (NB) is a solid tumor formed by the malignant proliferation of undifferentiated and immature neuroblasts. It often occurs in the neural crest of the extracranial sympathetic nervous system and is a common malignant tumor in children, accounting for 10% to 15% of childhood cancer-related mortality, seriously threatening the lives and health of minors. NB is characterized by significant heterogeneity in clinical outcomes, high incidence, rapid disease progression, high mortality, and great treatment difficulty, so it is often called "the king of childhood tumors".

[0003] The significant heterogeneity in clinical outcomes is an important feature of NB. The prognosis of high-risk neuroblastoma is very poor. Even with intensive comprehensive treatment, disease progression or even death still occurs, and the 5-year survival rate is only about 50%. The traditional treatments for NB include surgery, chemotherapy, and radiotherapy. The surgical treatment effect for low-risk patients is relatively good, and chemotherapy is required for intermediate-risk or advanced NB. Despite the application of the best treatment combination, more than 50% of high-risk NB patients still relapse, and about 40% of patients ultimately die, often due to multi-drug resistance and the difficulty of completely clearing minimal residual disease. Immunotherapy is hailed as the third revolutionary breakthrough in tumor treatment after radiotherapy, chemotherapy, and targeted therapy. This treatment strategy of mobilizing the patient's own immune resistance to eliminate tumors has become a new breakthrough point in tumor treatment. Immunotherapy mainly based on anti-GD2 monoclonal antibody (dinutuximab) has been included in the first-line treatment of NB. However, due to intensive treatment, patients often have severe complications, greatly reducing their quality of life. Therefore, it is of great significance to actively search for new drugs and their action targets for the treatment of NB.

[0004] In recent years, studies have shown that the tumor microenvironment (TME) plays a crucial role in the occurrence and development of NB. Tumor cells and non-tumor components can interact with each other by releasing secreted factors, cytokines, chemokines, or other means, resulting in immune escape and promoting tumor progression. Currently, targeting TME for tumor treatment has become a research hotspot related to NB. By regulating the cell composition, signaling pathways, and related molecules in TME, changing its immune infiltration characteristics, angiogenesis, extracellular matrix components, and metabolism, etc., the purpose of inhibiting tumor growth, diffusion, and metastasis can be achieved. Dimethyl succinate (DMS) is an esterification derivative of succinic acid. DMS can be hydrolyzed by esterase in vivo to generate succinic acid. Metabolomics analysis shows that DMS treatment leads to significant changes in the levels of TCA cycle intermediates, but the role of DMS in tumor progression is not yet clear. Bergapten (Bergamottin Bergapten (BGM) is a naturally occurring furocoumarin compound, mainly derived from citrus plants, especially the peel of Citrus bergamia. It is known to inhibit the growth of tumor cells. However, there is currently no evidence that bergapten can target and inhibit cancer metastasis, especially the epithelial-mesenchymal transition (EMT) process in malignant cells.

[0005] The patent application document with the publication number CN109864988A discloses the application of bergapten and / or vitexicarpin and products using the same. It provides the application of bergapten and / or vitexicarpin in the preparation of products for preventing and / or treating arenaviridae virus infections. Both bergapten and vitexicarpin are monomeric small molecule compounds of traditional Chinese medicine, which can effectively inhibit the infection of arenaviridae virus to cells within the non-toxic range, filling the current gap in the treatment of arenaviridae virus infections. The products prepared with bergapten and / or vitexicarpin as active ingredients include drugs and functional foods, which have good effects on the prevention and treatment of arenaviridae virus infections.

[0006] The patent application document with the publication number CN104173335A discloses the application of bergapten in the preparation of drugs for preventing and treating diabetes and in the preparation of functional foods or health products for preventing and treating diabetes. The chemical formula of the bergapten is C 21 H 22 O 4 , with a relative molecular mass of 338.4. The research of the present invention proves that bergapten shows significant glucose consumption activity in human hepatoma HepG2 cells, with good concentration dependence, and its action concentration is much lower than that of the positive drug metformin, while the drug effect is better than that of metformin. In diabetic KK-Ay mice, bergapten can significantly reduce the blood glucose of diabetic KK-Ay mice, increase glucose tolerance and insulin sensitivity. Through the hypoglycemic activity research of bergapten at the cellular and in vivo levels, the present invention shows that bergapten exhibits significant hypoglycemic activity, increases glucose tolerance and insulin sensitivity, and can be used as a therapeutic drug or functional food and health product for the prevention and treatment of diseases related to abnormal glucose metabolism.

[0007] The patent application document with the publication number CN116327757A discloses the use of bergapten and / or its pharmaceutically acceptable salts in the preparation of drugs for relieving, adjuvantly treating or treating tumors. Through research, it is found that in cell experiments, bergapten and / or its pharmaceutically acceptable salts can significantly inhibit the proliferation of tumor cells, significantly weaken the migration ability of tumor cells, significantly block the tumor cell cycle, and promote tumor cell apoptosis; in animal experiments, bergapten and / or its pharmaceutically acceptable salts can significantly inhibit the growth of tumor volume in a tumor-bearing animal model and reduce the increase in tumor mass; in summary, bergapten and / or its pharmaceutically acceptable salts can be used to prepare drugs for relieving, adjuvantly treating or treating tumors, especially in the treatment of glioblastoma.

[0008] There is currently no research report on the treatment of neuroblastoma with bergapten. Summary of the Invention

[0009] The present invention provides an application of bergapten in the preparation of drugs for treating neuroblastoma, and for the first time reveals the inhibitory effect of bergapten on neuroblastoma.

[0010] One of the technical solutions of the present invention is achieved by the following measures: an application of bergapten in the preparation of drugs for treating neuroblastoma.

[0011] Another technical solution of the present invention is achieved by the following measures: an application of bergapten in the preparation of drugs for treating nerve cell-derived tumors.

[0012] The following is a further optimization or / and improvement of the above-mentioned invention technical solution: The dosage form of the above drug is one or more of injection, tablet, capsule, powder and granule.

[0013] The present invention for the first time reveals the inhibitory effect of bergapten on neuroblastoma, and also for the first time discloses the application of bergapten targeting PLOD2 to exert anti-tumor effects. Through the pharmacodynamic experimental data, it can be seen that BGM inhibits tumor growth and inhibits the metastasis of neuroblastoma by targeting PLOD2 expression, indicating that bergapten can be used as a potential targeted anti-NB drug, thus providing a new target for the treatment of neuroblastoma. Brief Description of the Drawings

[0014] Appendix Figure 1-1 、 1-2 DMS promotes the migration, invasion, cell proliferation, EMT and liver metastasis of neuroblastoma.

[0015] Figure 1-1 、 1-2In (A), immunofluorescence (IF) staining of SH-SY5Y and LAN-5 cells treated with DMS (10 mM) for 1 day and 3 days; In (B), western blot analysis of the expression of E-cadherin, N-cadherin, and vimentin in SH-SY5Y and LAN-5 cells treated with DMS (10 mM); In (C), CCK-8 assay of SH-SY5Y and LAN-5 cells treated with DMS to measure cell viability; In (D), Transwell migration and invasion assays of SH-SY5Y and LAN-5 cells treated with DMS; In (E), H&E staining of mouse liver tissues injected with Neuro-2a cells and treated with DMS. *P<0.05, **P<0.01, ***P<0.001 compared with the control group.

[0016] Supplementary Figure 2-1 、 2-2 PLOD2 is crucial for succinate (DMS)-induced epithelial-mesenchymal transition (EMT) and cell migration.

[0017] Figure 2-1 、 2-2 In (A), heatmap of gene expression changes in SH-SY5Y and LAN-5 cells after DMS treatment; In (B), qRT-PCR and western blot analysis of PLOD2 expression in SH-SY5Y and LAN-5 cells transfected with shPLOD2-1 / 2 or negative control (shNC); In (C), CCK-8 assay of the viability of SH-SY5Y and LAN-5 cells after PLOD2 knockdown; In (D), Transwell migration and invasion assays of SH-SY5Y and LAN-5 cells after PLOD2 knockdown; In (E), western blot analysis of EMT markers (E-cadherin, N-cadherin, and vimentin) in SH-SY5Y and LAN-5 cells with PLOD2 knockdown; In (F), measurement of succinate levels in SH-SY5Y and LAN-5 cells with PLOD2 knockdown using a succinate detection kit. *P<0.05, **P<0.01, ***P<0.001.

[0018] Supplementary Figure 3-1 、 3-2, 3-3 shows that BGM inhibits the viability of neuroblastoma cells, as well as PLOD2 expression and the EMT process.

[0019] Figure 3-1 , 3-2 , In 3-3, (A) qRT-PCR analysis of PLOD2 mRNA levels in SH-SY5Y and LAN-5 cells treated with BGM (10 μm, 30 μm); (B) CCK-8 assay to determine the cell viability of SH-SY5Y and LAN-5 cells; (C) BGM significantly inhibits the succinate level in SH-SY5Y and LAN-5 cells; (D) qRT-PCR analysis of PLOD2 mRNA levels in SH-SY5Y and LAN-5 cells after PLOD2 overexpression and BGM treatment; (E) Detection of the proliferative viability of SH-SY5Y and LAN-5 cells treated with BGM combined with PLOD2 overexpression and BGM; (F) Detection of the succinate secretion level of SH-SY5Y and LAN-5 cells treated with BGM combined with PLOD2 overexpression and BGM; (G) Migration and invasion assays of SH-SY5Y and LAN-5 cells treated with BGM combined with PLOD2 overexpression and BGM; (H) Detection of the protein expression levels of EMT markers (E-cadherin, N-cadherin, Vimentin) in SH-SY5Y and LAN-5 cells treated with BGM combined with PLOD2 overexpression and BGM; * p<0.05, ** p<0.01, *** p<0.001.

[0020] Appendix Figure 4-1 , 4-2 shows that BGM inhibits tumor growth in neuroblastoma-bearing mice, promotes an increase in the number of CD8+ T cells, and reverses CD8+ T cell activation.

[0021] Figure 4-1 In, (A) In the Neuro-2a mouse model treated with BGM combined with PLOD2 overexpression / knockdown and BGM, it was observed that BGM significantly inhibited tumor growth, and the effect of the BGM combined with PLOD2 knockdown group was particularly significant; (B-D) In the Neuro-2a mouse model treated with BGM combined with PLOD2 overexpression / knockdown and BGM, BGM significantly inhibited the proportion of CD4 + T cells, promoted a significant increase in CD8 + T cells, and the effect of the BGM combined with PLOD2 knockdown group was particularly significant; Figure 4-2In the BGM and α-CD8 combined with BGM-treated Neuro-2a mouse model, BGM can significantly inhibit tumor growth and the EMT process. Detailed implementation mode

[0022] The present invention is not limited by the following embodiments, and the specific implementation mode can be determined according to the technical solution of the present invention and the actual situation. Various chemical reagents and chemical supplies mentioned in the present invention are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified. The present invention will be further described below with reference to the embodiments: Example 1: Application of bergapten in the preparation of drugs for treating neuroblastoma.

[0023] Example 2: Application of bergapten in the preparation of drugs for treating neurocyte-derived tumors.

[0024] Example 3: As an optimization of the above embodiments, the dosage form of the drug is one or more of injection, tablet, capsule, powder and granule.

[0025] The following is the specific pharmacodynamic experiment of bergapten on neuroblastoma described in the present invention: 1. Experimental content Cell culture and treatment The human neuroblastoma cell lines SH-SY5Y (female) and LAN-5 (male) were obtained from ATCC (American Type Culture Collection) and cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1% glutamine at 37°C and 5% CO 2 2. The cells were treated with dimethyl succinate (DMS; 10 mM) or bergapten (BGM; 10 μM, 30 μM) for 1 day to 3 days, with DMSO as the vehicle control.

[0026] Cell transfection SH-SY5Y and LAN-5 cells were transfected with siRNA targeting IGF2BP3 or scrambled siRNA (siNC), and shRNA targeting PLOD2 or scrambled shRNA (shNC) using Lipofectamine 3000. Stable knockdown cells were selected with puromycin. For PLOD2 overexpression, cells were transfected with pcDNA3.1-PLOD2 plasmid or empty vector. The transfection efficiency was verified by qRT-PCR and Western blot. In vivo, CD8 + T cells were depleted by intraperitoneal injection of αCD8 antibody (200 μg / mouse) every 3 days. Flow cytometry confirmed the successful depletion of CD8 + T cells, ensuring the functional verification of T cell-mediated effects.

[0027] Western Blot Experiment Proteins were extracted using RIPA buffer containing protease and phosphatase inhibitors (Sigma - Aldrich). Equal amounts of proteins were separated by SDS - PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non - fat milk and incubated overnight at 4°C with primary antibodies against PLOD2 (1:1000, ab314640, Abcam), IGF2BP3 (1:1000, ab289247, Abcam), E - cadherin (1:1000, #14472, Cell Signaling Technology), N - cadherin (1:5000, ab76011, Abcam) and Vimentin (1:1000, #5741, Cell Signaling Technology), followed by HRP - conjugated secondary antibodies. Protein bands were visualized using an ECL detection system (Thermo Fisher).

[0028] Real - Time Fluorescent Quantitative PCR (qRT - PCR) Experiment Total RNA was extracted from SH - SY5Y and LAN - 5 cells using TRIzol reagent (Invitrogen) and reverse - transcribed using the PrimeScript RT kit (Takara). Quantitative PCR was performed using SYBR Green premix (Applied Biosystems) on a QuantStudio 5 system (Thermo Fisher). The relative gene expression of PLOD2, LOX, LOX1, LOX2, PHYH, P4HA3, ASPHD1, YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, IGF2BP1, IGF2BP2, IGF2BP3, HNRNPC, HNRNPG, PLOD2 - related m6A and the housekeeping gene GAPDH was calculated using the 2−ΔΔCt method.

[0029] Cell Proliferation Detection (CCK - 8) The Cell Counting Kit - 8 (CCK - 8; Tongrentang) was used. SH - SY5Y and LAN - 5 cells were seeded at 3×10 3Cells were seeded at a density of cells / well in 100 μL of complete medium. After 24 hours, the cells were treated with BGM (10 μM, 30 μM) or vehicle control (DMSO). At 24 hours, 48 hours, and 72 hours after treatment, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 hours. Absorbance at 450 nm was measured using a microplate reader (BioTek). The proliferation rate was calculated by normalizing the absorbance values to the control group. Each experiment was performed in triplicate.

[0030] Observation of liver metastasis A tail vein injection model using C57BL / 6 mice was used to evaluate liver metastasis. Neuro-2a cells were suspended in PBS at a concentration of 2×10 6 cells / mL. Mice were injected via the lateral tail vein with 1×10 6 cells (50 μL) and treated every other day with BGM (1 mg / kg, intraperitoneal) or vehicle (DMSO) for 14 days. At the end point, the livers were harvested, fixed with 4% paraformaldehyde, and processed for histological analysis. H&E staining was used to observe metastatic nodules, and immunohistochemical staining was performed to detect EMT markers, including E-cadherin, N-cadherin, and PLOD2. The number and size of metastatic nodules.

[0031] Immunofluorescence (IF) staining SH-SY5Y and LAN-5 cells were seeded on coverslips, fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.1% Triton X-100. After blocking with 5% BSA for 30 minutes, the cells were incubated with primary antibodies against E-cadherin, N-cadherin, vimentin, PLOD2, and IGF2BP3 overnight at 4 °C. After washing, the samples were incubated with fluorescently labeled secondary antibodies for 1 hour. Nuclei were stained with DAPI, and images were captured using a confocal fluorescence microscope (Leica).

[0032] Succinate quantification Succinate levels in treated SH-SY5Y and LAN-5 cells were measured using a succinate detection kit (Abcam, ab204718). Cell lysates were prepared and incubated with the reagents of the kit according to the manufacturer's protocol. Absorbance was measured at 450 nm using a microplate reader. Succinate levels were calculated according to the standard curve.

[0033] Migration and invasion assays Transwell chambers (8 μm pore size, Corning) were used to evaluate the migration and invasion of SH-SY5Y and LAN-5 cells. For the migration assay, cells (2×10 4(0) Inoculate in serum-free medium in the upper chamber, while adding 10% FBS to the lower chamber. For the invasion assay, the upper chamber is pre-coated with Matrigel (BD Biosciences). After 24 hours, fix the cells that have migrated or invaded on the lower membrane, stain with crystal violet, and count under an optical microscope.

[0034] CD8 + CD8 T cell isolation and chemotaxis assay Isolate CD8 T cells from the spleens of 4-week-old male C57BL / 6 mice using magnetic-activated cell sorting (MACS, Miltenyi Biotec). For the chemotaxis assay, place the conditioned medium from treated SH-SY5Y and LAN-5 neuroblastoma cells in the lower chamber of a Transwell plate, and add 5×10 + CD8 5 CD8 + T cells to the upper chamber. After 6 hours, count the CD8 T cells that have migrated to the lower chamber under a microscope. + T cells.

[0035] In vivo tumor model Subcutaneously inject 4×10 4-week-old male C57BL / 6 mice with Neuro-2a neuroblastoma cells into the shoulder region. Randomly divide the mice into the following groups: (1) control (vehicle-treated), (2) BGM (1 mg / kg, intraperitoneal, once every other day), (3) BGM + PLOD2 (BGM with tumor-specific PLOD2 overexpression), (4) BGM + shPLOD2 (BGM with PLOD2 knockdown), (5) αCD8 (CD8 T cell depletion with αCD8 antibody, 200 μg / mouse every 3 days), and (6) αCD8 + BGM (CD8 T cell depletion combined with BGM treatment). Monitor tumor volume and weight, and analyze CD8 T cell infiltration, PLOD2 expression, and EMT markers (E-cadherin, N-cadherin, vimentin) of the harvested tumors by IHC. Flow cytometry confirms CD8 T cell depletion. 6 + T cells + T cells + T cell infiltration, PLOD2 expression, and EMT markers (E-cadherin, N-cadherin, vimentin). Flow cytometry confirms CD8 + T cell depletion.

[0036] Immunohistochemistry For immunohistochemistry (IHC), tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-μm slices. After dewaxing and rehydration, antigen retrieval was performed using citrate buffer (pH 6.0). The sections were blocked with 5% BSA and incubated overnight at 4°C with primary antibodies against PLOD2 (1:2000, ab314640, Abcam), CD8 (1:2000, ab217344, Abcam), CD4 (1:500, ab133616, Abcam), E-cadherin (1:500, #14472, Cell Signaling Technology), N-cadherin (1:5000, ab76011, Abcam), and Vimentin (1:200, #5741, Cell Signaling Technology). After washing, HRP-conjugated secondary antibodies and DAB substrate were applied for visualization. Hematoxylin was used for nuclear counterstaining.

[0037] Molecular docking analysis Molecular docking was performed using AutoDock Vina to evaluate the binding of BGM to PLOD2 and IGF2BP3. Protein structures were retrieved from the Protein Data Bank (PDB) or predicted by homology modeling. The ligand (BGM) structure was optimized using the MMFF94 force field. Protein-ligand interactions, binding sites, and binding affinities (kcal / mol) were analyzed and visualized using PyMOL. Key residues involved in hydrogen bonding and hydrophobic interactions were identified.

[0038] m6A RNA immunoprecipitation (RIP) and m6A RIP-qPCR m6A-modified RNAs were enriched using the Magna MeRIP m6A kit (Millipore). After immunoprecipitation of total RNA with anti-m6A antibody or IgG (negative control), the m6A enrichment on PLOD2 mRNA was determined by qRT-PCR. The relative enrichment was normalized to the input RNA.

[0039] Result analysis To investigate the role of DMS in neuroblastoma progression, the expression of epithelial-mesenchymal transition (EMT)-related proteins and cell behaviors were examined in SH-SY5Y and LAN-5 cell lines. Immunofluorescence IF staining showed that DMS treatment significantly decreased E-cadherin expression, while increasing N-cadherin and vimentin levels in both cell lines over time (1d and 3d), indicating that DMS induced EMT in neuroblastoma cells (Figure 1A). Western blot analysis confirmed these findings, with a significant decrease in E-cadherin and an increase in N-cadherin and vimentin levels after DMS treatment (Figure 1B). To evaluate the effect of DMS on proliferation, CCK-8 results showed a significant increase in cell viability of SH-SY5Y and LAN-5 cells after DMS treatment (Figure 1C). In addition, Transwell assay results showed that DMS treatment enhanced cell migration and invasion, as evidenced by a significant increase in the number of migrated and invaded cells (Figure 1D). In the liver metastasis model, H&E staining of liver tissues showed that mice treated with DMS exhibited a significant increase in the number and size of metastatic nodules compared to the control group (Figure 1E).

[0040] To identify the key metabolic regulators involved in DMS-induced EMT, the expression of α-KG-dependent dioxygenases and related metabolic genes (including SDH) were analyzed. Heatmap analysis identified PLOD2 as the most significantly upregulated gene in DMS-treated SH-SY5Y and LAN-5 cells, indicating its role in the EMT process (Figure 2A). To confirm the functional role of PLOD2, PLOD2 knockdown was performed using two independent shRNAs (shPLOD2-1 and shPLOD2-2). Effective silencing of PLOD2 expression was verified by qRT-PCR and Western blot analysis (Figure 2B). Determined by CCK-8 assay, knockdown of PLOD2 significantly decreased cell proliferation (Figure 2C). In addition, Transwell assay showed a significant reduction in cell migration and invasion after PLOD2 silencing (Figure 2D). The effect of PLOD2 knockdown on EMT markers was evaluated by Western blot analysis, and the results showed a restoration of E-cadherin expression and inhibition of N-cadherin and vimentin levels in SH-SY5Y and LAN-5 cells (Figure 2E). In addition, the succinate level in PLOD2-silenced neuroblastoma cells was significantly decreased as determined by microplate reader (Figure 2F). These findings indicate that PLOD2 plays a key role in mediating DMS-induced EMT and cell metabolism in neuroblastoma cells, positioning it as a potential therapeutic target in this context.

[0041] To determine whether BGM exerts antitumor effects by targeting PLOD2, its effects on PLOD2 expression, cell proliferation, and succinate levels in SH-SY5Y and LAN-5 cells were investigated. qRT-PCR analysis showed that BGM treatment significantly reduced the mRNA level of PLOD2 in a dose-dependent manner, with the strongest inhibitory effect at 30 μM (Figure 3A). PLOD2 overexpression restored the mRNA expression level after BGM treatment, confirming the specificity of the regulatory effect of BGM (Figure 3D). To evaluate the effect of BGM on cell proliferation, a CCK-8 assay was performed. BGM treatment significantly decreased the viability of SH-SY5Y and LAN-5 cells in a dose-dependent manner (Figure 3B). PLOD2 overexpression rescued the viability decrease induced by BGM, while PLOD2 silencing further enhanced the antiproliferative effect of BGM (Figure 3E). To evaluate the effect of BGM on succinate metabolism, intracellular succinate levels were measured. BGM treatment significantly decreased succinate levels in a dose-dependent manner (Figure 3C), indicating inhibition of the metabolic process associated with PLOD2. PLOD2 overexpression restored the succinate levels inhibited by BGM, while PLOD2 knockdown further decreased succinate levels, supporting the key role of PLOD2 in succinate regulation (Figure 3F). Transwell assays showed that BGM significantly inhibited the migration and invasion abilities of SH-SY5Y and LAN-5 cells (Figure 3G). PLOD2 overexpression partially reversed these effects and restored the migration and invasion abilities, while PLOD2 silencing further inhibited these processes, indicating that the inhibition of migration and invasion by BGM is mediated by PLOD2 regulation. The effect of BGM on EMT markers was evaluated using Western blot analysis. BGM treatment increased the expression of E-cadherin (epithelial marker) and decreased the levels of N-cadherin and vimentin (mesenchymal markers), indicating inhibition of EMT (Figure 3H). PLOD2 overexpression reversed these changes and restored the mesenchymal phenotype, while PLOD2 silencing further enhanced the inhibitory effect of BGM on EMT markers. These findings demonstrated that BGM inhibits the proliferation, migration, invasion, and EMT of neuroblastoma cells by targeting PLOD2. The results of overexpression and silencing experiments further confirmed the central role of PLOD2 in mediating these effects, particularly through its regulation of metabolic activities and EMT-related processes.

[0042] To evaluate the in vivo antitumor effect of BGM and its impact on the tumor immune microenvironment, a subcutaneous neuroblastoma xenograft model was established and CD8 was analyzed +T cell infiltration. Tumor growth analysis showed that compared with the control group, BGM treatment significantly reduced tumor volume and weight. PLOD2 overexpression partially restored tumor growth, while PLOD2 silencing enhanced the tumor-suppressive effect of BGM, indicating that PLOD2 mediates the anti-tumor activity of BGM (Figure 4A). To examine the tumor immune microenvironment, flow cytometry was performed to evaluate CD8 + T and CD4 + T cell infiltration. BGM treatment significantly increased CD8 + T cell infiltration, while the CD4+ T cell level remained unchanged. PLOD2 overexpression reversed the increase in CD8 + T cell infiltration, while PLOD2 silencing amplified the effect of BGM, indicating that PLOD2 inhibits CD8 + T cell-mediated anti-tumor immunity (Figure 4B). Immunohistochemistry (IHC) analysis further confirmed these findings. BGM treatment reduced PLOD2 expression and increased CD8 + T cell infiltration in tumor tissues. PLOD2 overexpression restored PLOD2 levels and reduced the number of CD8 + T cells, while PLOD2 silencing enhanced CD8 + T cell infiltration (Figure 4C). Quantification of CD8 + T cell numbers showed a significant increase in infiltration after BGM treatment, which was reversed by PLOD2 overexpression and amplified by PLOD2 silencing (Figure 4D). To confirm the role of CD8 + T cells in the anti-tumor effect of BGM, CD8 + T cell depletion was performed using an αCD8 antibody. Depletion of CD8 + T cells abolished the tumor-suppressive effect of BGM, as demonstrated by the increased tumor volume and weight in the αCD8-treated group compared with the BGM-treated group (Figure 4E). IHC analysis of EMT markers showed that BGM treatment increased E-cadherin expression while decreasing N-cadherin and vimentin levels, indicating inhibition of EMT. Depletion of CD8 + T cells reversed these changes, resulting in decreased E-cadherin and increased N-cadherin and vimentin levels (Figure 4F). This indicates that CD8 + T cells mediate the inhibition of EMT by BGM.

[0043] From the above pharmacodynamic experimental data, it can be seen that BGM inhibits tumor growth by targeting PLOD2 expression and inhibits neuroblastoma metastasis, indicating that bergapten can be used as a potential drug targeting anti-NB, thus providing a new target for the treatment of neuroblastoma.

[0044] The above technical features respectively constitute the embodiments of the present invention, which have strong adaptability and implementation effects. Non-essential technical features can be increased or decreased according to actual needs to meet the requirements of different situations.

Claims

1. An application of bergamot in the preparation of a drug for treating neuroblastoma.

2. An application of bergamot in the preparation of drugs for treating neuronal cell-derived tumors.

3. The use according to claim 1 or 2, characterized in that: The dosage form of the drug is one or more of injection, tablet, capsule, powder and granule.

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