Application of berberrubine in preparation of medicine for preventing and treating lung cancer

Abstract

The invention provides application of berberrubine in preparation of a medicine for preventing and treating lung cancer. The technical scheme provided by the invention is developed around an MDM2 / p53 pathway, tumor inhibition is realized by inducing cell aging, and the technical scheme is clear in molecular mechanism and definite in intervention target, and has repeatability and verifiability. Compared with traditional radiotherapy and chemotherapy drugs, the technical scheme provided by the invention does not cause acute cell death, and the overall toxicity is relatively low. Meanwhile, according to the technical scheme provided by the invention, the preparation process of the raw material berberrubine is mature, and the berberrubine can be developed into various dosage forms and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of berberine red in the preparation of a drug for preventing and treating lung cancer. BACKGROUND

[0002] Lung cancer is one of the highest incidence and mortality rates of malignant tumors in the world, and there are still problems such as high recurrence rate, high metastasis and drug resistance. Traditional treatment methods such as surgery, radiotherapy and targeted therapy, mainly focus on directly killing tumor cells, but are prone to produce toxic side effects, and cannot continuously inhibit tumor progression.

[0003] At present, the treatment of malignant tumors in clinical practice mainly includes surgical resection, chemotherapy and radiotherapy, but surgical treatment can only achieve radical cure for a small part of early cancer patients, and most cancer patients are diagnosed at an advanced stage, and most lung cancer patients have no opportunity for surgery. Chemotherapy has great side effects, and the quality of life of lung cancer patients is poor. Radiotherapy is a local treatment method, but it has poor effect on distant metastatic tumors, and also has great side effects. Radiotherapy, chemotherapy and targeted drugs can kill tumor cells, but have limited regulation on tumor-related vascular systems, and may even promote tumor escape and recurrence. At the same time, drug resistance greatly reduces the treatment effect. Based on the limitations of the above treatment methods, it is particularly important to find new effective anti-malignant tumor drugs.

[0004] In recent years, it has been found that cell senescence as a non-apoptotic permanent cell cycle arrest state has gradually become a new anti-tumor strategy. Inducing tumor cell senescence can effectively inhibit its proliferation and clonogenic ability, and achieve tumor growth control without relying on cytotoxicity. Especially in some strong drug resistance and active division of malignant tumor types, induction of senescence intervention is considered a safe and sustainable treatment idea. Studies have found that imidazoline compounds, piperidine compounds and other compounds also have the ability to activate the P53 pathway and induce cell senescence, which can regulate key molecules such as MDM2 / p53 to make tumor cells enter the cell cycle arrest and senescence state.

[0005] Traditional Chinese medicine has the advantages of multi-target and low toxicity in inducing tumor cell senescence. The main component of Huanglian, berberine (BRB), has been widely studied and confirmed to have biological activities such as anti-proliferation and induction of cell apoptosis. The patent document with the publication number CN114195779A discloses the application of 8-alkyl berberine salt in the preparation of drugs for preventing and treating lung cancer, and provides a new use of long-chain 8-alkyl berberine salt in the prevention and treatment of lung cancer. Especially when the alkyl chain carbon number of the compound is 12-22, the anti-lung cancer effect is significantly better than that of berberine hydrochloride salt, and it can be used for preparing drugs for preventing and treating lung cancer. However, the specific mechanism of berberine in inducing senescence in lung cancer cells, especially its role in ubiquitin regulation, P53 activation, and cell cycle regulation, still needs further research and systematic interpretation. Based on the above reasons, the application potential of berberine in the preparation of lung cancer antitumor drugs has not been fully explored. SUMMARY

[0006] The purpose of the present application is to provide the application of berberine in the preparation of drugs for preventing and treating lung cancer.

[0007] Another purpose of the present application is to provide the application of berberine in the preparation of MDM2 inhibitors.

[0008] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0009] The application of berberine in the preparation of drugs for preventing and treating lung cancer, wherein the lung cancer is non-small cell lung cancer expressing wild-type p53 protein. In an embodiment of the present application, the non-small cell lung cancer expressing wild-type p53 protein is specifically A549 cells and H460 cells.

[0010] Preferably, the lung cancer drug inhibits the protein interaction between MDM2 and p53, prolongs the half-life of p53 protein, and induces tumor cell senescence. In an embodiment of the present application, MST experiments verify that berberine can specifically bind to MDM2; SPR experiments verify that BRB has sub-micromolar binding affinity with MDM2 protein, and the measured dissociation constant (3.263 μM) indicates that BRB can form a stable complex with MDM2; Co-Immunoprecipitation experiments and Western Blot experiments verify that BRB can significantly inhibit the ubiquitination modification level of p53 protein Figure 16 、 Figure 17 ), can significantly delay the degradation rate of p53 protein (BRB can prolong the degradation time of p53 protein by about 8 times according to Figure 13 、 15 ), and prolong its half-life.

[0011] Preferably, the lung cancer drug is capable of inhibiting the growth of A549 subcutaneous transplanted tumor in an animal model. In one embodiment of the present application, the tumor inhibition rate of the 100 mg / kg BRB treatment group and the 150 mg / kg BRB treatment group is 40.6% and 53.6% respectively, compared with the control group (here, the tumor inhibition rate is the value of (tumor volume of the control group - tumor volume of the BRB treatment group) / tumor volume of the control group at the last measurement of the tumor volume).

[0012] Preferably, the lung cancer drug is a pharmaceutical composition comprising:

[0013] a. berberrubine or a pharmaceutically acceptable salt thereof; and b. a pharmaceutically acceptable carrier.

[0014] Further preferably, the carrier comprises at least one of microspheres, liposomes, nanoparticles, and microemulsions.

[0015] The use of berberrubine in the preparation of an MDM2 inhibitor capable of specifically binding to MDM2 protein, inhibiting the ubiquitination and degradation of p53 protein, and up-regulating the expression level of P21 mRNA in non-small cell lung cancer expressing wild-type p53 protein. In one embodiment of the present application, it is verified by Co-Immunoprecipitation experiment and Western Blot experiment that BRB can significantly inhibit the ubiquitination modification level of p53 protein (according to Figure 16 , 17 ), significantly delay the degradation rate of p53 protein (according to Figure 13 , 15 , BRB can prolong the degradation time of p53 protein by about 8 times), and prolong its half-life; it is verified by real-time fluorescent quantitative PCR experiment that BRB can significantly up-regulate the P21 mRNA level (according to Figure 11 , up-regulate the A549 cells by 9.3 times; up-regulate the H460 cells by 2.9 times);

[0016] Preferably, the MDM2 inhibitor is a pharmaceutical composition comprising:

[0017] a. berberrubine or a pharmaceutically acceptable salt thereof; and b. a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the above technical solution has the following beneficial effects:

[0019] This invention provides an application of berberine in the preparation of drugs for the prevention and treatment of lung cancer. The technical solution provided by this invention revolves around the MDM2 / p53 pathway, achieving tumor suppression by inducing cellular senescence. This technical solution has a clear molecular mechanism, a well-defined intervention target, and is reproducible and verifiable.

[0020] Compared to traditional radiotherapy and chemotherapy drugs, the technical solution provided by this invention does not cause acute cell death and has lower overall toxicity. Furthermore, the preparation process of berberine, the raw material for the technical solution provided by this invention, is mature and can be developed into various dosage forms, showing promising application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structural formula of berberine, the experimental drug used in the examples;

[0022] Figure 2 This is a schematic diagram of a pre-defined BRB molecular model;

[0023] Figure 3 This is a schematic diagram of the preset MDM2 molecular model;

[0024] Figure 4 A 3D schematic diagram and a magnified view of the simulated docking structure of BRB and MDM2 molecules;

[0025] Figure 5 A schematic diagram of docking to simulate the hydrophilicity / hydrophobicity display mode of molecular docking experiments;

[0026] Figure 6 Interaction diagrams for simulating molecular docking experiments in 2D display mode;

[0027] Figure 7 A schematic diagram of the molecular dynamics simulation test results for a simulated molecular docking experiment;

[0028] Figure 8 Figure showing the results of a micro-thermophoretic experiment involving BRB and MDM2;

[0029] Figure 9 Figure 1 shows the results of surface plasmon resonance experiments on BRB and MDM2.

[0030] Figure 10 A bar chart showing the relative expression levels of P53 mRNA in A549 and H460 lung cancer cells;

[0031] Figure 11 A bar chart showing the relative expression levels of P21 mRNA in A549 and H460 lung cancer cells;

[0032] Figure 12 Figure 1 shows the results of a Western blot experiment on p53 in H460 cells.

[0033] Figure 13 The graph shows the changes in p53 protein levels in H460 cells with CHX treatment at different time points, where the vertical axis represents the relative expression level of p53 protein.

[0034] Figure 14 Figure 1 shows the results of a Western blot experiment on p53 in A549 cells.

[0035] Figure 15 The graph shows the changes in p53 protein levels in A549 cells with CHX treatment at different time points, where the vertical axis represents the relative expression level of p53 protein.

[0036] Figure 16 This is a comparison of the ubiquitination modification levels in A549 and H460 lung cancer cells; Figure 16 -a represents A549 cells; Figure 16 -b represents H460 cells;

[0037] Figure 17 This is a comparison of the Co-IP and Western Blot results for A549 and H460 lung cancer cells; Figure 17 -a represents A549 cells; Figure 17 -b represents H460 cells;

[0038] Figure 18 The results of a Western blot experiment in A549 cells with P21 gene knockout are shown in the figure; WT represents A549 cells without P21 knockout; P21KO#1 and P21KO#2 are randomly selected A549 cells after P21 knockout.

[0039] Figure 19 The results of a Western blot experiment in which the P53 gene was knocked out in A549 cells; WT in the figure represents A549 cells without P53 knockout; P53KO represents randomly selected A549 cells after P53 knockout.

[0040] Figure 20 This is a diagram showing the results of one of the SA-β-gal staining experiments.

[0041] Figure 21 A bar chart showing the proportion of senescent A549 cells to all A549 cells in treatments with / without BRB addition and treatments with / without P21 gene knockout.

[0042] Figure 22 The image shows the experimental results of the second SA-β-gal staining experiment;

[0043] Figure 23 A bar chart showing the proportion of senescent A549 cells to all A549 cells in treatments with / without BRB addition and treatments with / without P53 gene knockout.

[0044] Figure 24 Figure 1 shows the results of immunofluorescence experiments on A549 and H460 lung cancer cells.

[0045] Figure 25 A bar chart showing the percentage of γ-H2AX positive cells in A549 and H460 lung cancer cells.

[0046] Figure 26 Western blotting results of Lamin B1 markers in A549 and H460 lung cancer cells;

[0047] Figure 27 Staining images of senescent cells after treatment with different concentrations of BRB in NSCLC lineages;

[0048] Figure 28 A bar chart showing the percentage of senescent cells after treating NSCLC lineages with different concentrations of BRB;

[0049] Figure 29 This is a graph showing the results of mouse weight change detection;

[0050] Figure 30 This is a graph showing the results of detecting changes in the volume of subcutaneous xenografts in mice.

[0051] Figure 31 Image showing the morphological results of subcutaneous xenograft tumors in mice;

[0052] Figure 32 Image showing the results of weighing subcutaneous xenografts in mice;

[0053] Figure 33 This is one of the images showing the results of HE staining experiments on mouse liver and kidneys;

[0054] Figure 34 Figure 2 shows the results of HE staining experiments on mouse liver and kidneys.

[0055] Figure 35 The image shows the results of ALT detection, a liver function indicator in mice.

[0056] Figure 36 The graph shows the results of AST (associated liver function) detection in mice.

[0057] Figure 37 The graph shows the results of UN (renal function) testing in mice.

[0058] Figure 38The graph shows the results of CREA test for renal function in mice.

[0059] Figure 39 The graph shows the results of the mouse liver weight / body weight test.

[0060] Figure 40 The graph shows the results of the mouse kidney weight / body weight test. Detailed Implementation

[0061] To illustrate in detail the technical problems, technical means, and technical effects of the technical solutions provided by this invention, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0062] The following text introduces experimental drugs and other technical terms:

[0063] Berberine, hereinafter referred to as BRB, has the following structural formula: Figure 1 As shown; the BRB used in the examples was purchased from Chengdu Glip Biotechnology Co., Ltd.; specification: 2g; CAS number: 15401-69-1.

[0064] Dimethyl sulfoxide (DMSO); CAS No.: 67-68-5;

[0065] Cycloheximide, hereinafter referred to as CHX;

[0066] Mouse Double Minute 2 Homolog (MDM2, hereinafter referred to as MDM2); purchased from Santa Cruz Biotechnology, Inc.; CAS No.: sc-965; specification: 200ug / ml;

[0067] Cyclin-dependent kinase inhibitor 1A (p21, hereinafter referred to as p21); purchased from Proteintech Group, Inc.; CAS No.: 10355-1-AP; specification: 100ul;

[0068] Tumor protein p53 (hereinafter referred to as p53); purchased from Proteintech Group, Inc.; CAS No.: 10442-1-AP; size: 100 μl;

[0069] Phosphorylated p53 protein, hereinafter referred to as p-p53;

[0070] Beta-actin monoclonal antibody (hereinafter referred to as β-actin) was purchased from Proteintech Group, Inc.; CAS No.: 66009-1-ig; 100ul.

[0071] Non-small cell lung cancer (NSCLC) lineage cells, hereinafter referred to as NSCLC lineage cells, include:

[0072] NCI-H1975 (human lung adenocarcinoma cells), hereinafter referred to as H1975 cells;

[0073] A549 (human lung adenocarcinoma cells), hereinafter referred to as A549 cells;

[0074] NCI-H2030 (human non-small cell lung cancer cells), hereinafter referred to as H2030 cells;

[0075] NCI-H1650 (human non-small cell lung cancer cells), hereinafter referred to as H1650 cells;

[0076] NCI-H460 (human large cell lung cancer cells), hereinafter referred to as H460 cells;

[0077] NCI-H1299 (human non-small cell lung cancer cells), hereinafter referred to as H1299 cells

[0078] And EKVX (human non-small cell lung cancer cells); all the NSCLC lineage cells mentioned above were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0079] Example 1: Introduction and Verification of the Antitumor Mechanism of Berberine

[0080] 1. Brief introduction to the mechanism of action:

[0081] This invention discloses the application of berberine in the preparation of drugs for the prevention and treatment of lung cancer; further, this invention discloses the application of berberine in the preparation of lung cancer cell inhibitors; the antitumor mechanism of berberine in inducing lung cancer cell senescence is described below:

[0082] Laboratory studies (described below) have confirmed that BRB can target and bind to MDM2. Once bound to MDM2, BRB inhibits the ubiquitination and degradation of p53 protein by MDM2, enhancing p53 protein stability and transcriptional activity. Furthermore, the binding of BRB to MDM2 upregulates key regulatory proteins such as p21 in downstream p53 pathways, ultimately inducing cell cycle arrest and senescence in lung cancer cells, thereby inhibiting tumor cell proliferation.

[0083] 2. Experimental validation of BRB-targeted binding to MDM2:

[0084] 2.1 Molecular dynamics simulation experiment:

[0085] (1) Parameter settings:

[0086] ①Preset BRB molecular model

[0087] The 3D structure of BRB (CAS: 15401-69-1) was downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The 3D structure of BRB was imported into Chem3D software to construct an initial structural model of berberine. Then, in Gaussian 16 software, the initial structure of BRB was optimized using the B3LYP functional of DFT under the 6-311g(d) basis set to obtain the optimized BRB molecular structure. Next, the optimized BRB structure was further processed using AutoDock Tools 1.5.7 software, including operations such as determining the root of the ligands and selecting ligand-twistable bonds, ultimately generating a pdbqt format file for docking. The final modeling illustration of the obtained BRB molecular model is shown below. Figure 2 .

[0088] ②Preset MDM2 molecular model

[0089] The structure file (7BIA) of MDM2 was downloaded from the PDB database (http: / / www.rcsb.org / ). The MDM2 structure file (7BIA) was then imported into software (PyMOL 2.5.5) for preprocessing. This preprocessing included, but was not limited to, removing redundant protein chains, water molecules, and other excess molecules. The preprocessed initial structure of MDM2 was then imported into another software (AutoDock Tools 1.5.7) for further processing—adding polarized hydrogen atoms, calculating charges, assigning atom types, etc.—to generate a pdbqt format file for simulating molecular docking. The final model of the MDM2 molecular model is shown below. Figure 3 .

[0090] (2) Molecular docking simulation:

[0091] Using AutoDockTools 1.5.7 software, the pdbqt format files of the aforementioned preset BRB molecular model and MDM2 molecular model were opened respectively. Docking was performed using a docking box, with 20 docking attempts per round, for a total of 3 rounds. The structure with the highest binding energy among all docking attempts was selected as the final docking result. The docking box was constructed using Gromacs 2023.3 software with dimensions of 7.571 × 7.749 × 7.418 nm. 3 The box.

[0092] (3) Display of molecular simulation docking results:

[0093] The docking results of MDM2 and BRB were visualized in PyMOL software. The simulated molecular docking experimental results are shown below. Figure 4 , Figure 5 , Figure 6 , Figure 7 As shown,

[0094] Figure 4 This is a 3D schematic diagram of the simulated docking structure of BRB and MDM2 molecules; the dashed boxes represent local binding details of the BRB and MDM2 molecule docking; according to Figure 4 It can be seen that MDM2 and BRB underwent molecular docking, and the binding energy of the active binding site of MDM2 and BRB is -7.504 kcal / mol.

[0095] Figure 5 A schematic diagram of docking to simulate the hydrophilicity / hydrophobicity display mode of molecular docking experiments; based on Figure 5 It can be seen that BRB binds in the protein hydrophobic pocket of MDM2;

[0096] Figure 6 Interaction diagrams for 2D visualization of molecular docking experiments; based on Figure 6 It can be seen that BRB forms a hydrogen bond with the L54 amino acid residue of MDM2, and exhibits van der Waals hydrophobic interactions with 10 amino acid residues, including L57, G58, and I61. L54, L57, G58, and I61 are all amino acid residues.

[0097] Figure 7 This is a schematic diagram of the molecular dynamics simulation test results for a simulated molecular docking experiment; based on Figure 7 It can be seen that after MDM2 and BRB form a complex, its RMSD value remains stable after 50 ns. This indicates that MDM2 and BRB can stably bind within the binding domain, and this binding competes with the binding of p53 to MDM2.

[0098] Based on the above simulation experiments, it can be concluded that under the simulated conditions, BRB and MDM2 can spontaneously bind and exhibit strong binding.

[0099] 2.2 Micro-thermophoresis experiment

[0100] The experimental reagents BRB and MDM2 were subjected to microscale thermophoresis (MST) to detect the binding affinity between BRB and MDM2. The MST experimental procedure is shown below:

[0101] Step 1: Preparation of dye working solution

[0102] Take 3.6 μL of MST-specific fluorescent dye and mix it with 176.4 μL of PBST buffer to prepare a dye working solution with a final concentration of 100 nM.

[0103] Step 2: Protein Sample Processing

[0104] The purified MDM2 protein was diluted to 200 nM with PBST buffer to obtain a diluted MDM2 protein solution, which was then placed on ice for later use.

[0105] Step 3: Formation of the protein-dye complex

[0106] Mix the dye working solution prepared in step 1 with the diluted MDM2 protein solution obtained in step 2, and mix by pipetting and aspirating in equal volumes. Incubate at 25±1℃ for 30 minutes under dark conditions to form a fluorescently labeled protein complex.

[0107] Step 4: Gradual Dilution of Drug

[0108] Using PBST buffer as a diluent, the BRB compound stock solution was serially diluted to prepare 13 concentration gradient drug solutions in 200 μL centrifuge tubes. The concentrations were: 0.061 μM, 0.122 μM, 0.244 μM, 0.488 μM, 0.977 μM, 1.953 μM, 3.906 μM, 7.813 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, and 250 μM.

[0109] Step 5: Construction of the reaction system

[0110] Take 5 μL of the fluorescently labeled protein complex prepared in step 3, and mix it with an equal volume of the BRB drug solutions of each concentration obtained in step 4. Mix thoroughly by pipetting. Transfer the mixed solution to the corresponding wells of a 384-well black microplate.

[0111] Step 6: Detection and Data Analysis

[0112] The 384-well microplate was centrifuged at 4000 rpm for 2 minutes at 4°C to remove air bubbles. It was then placed in a micro-thermophoresis apparatus for signal acquisition at 25°C. The dose-response curves were fitted using the instrument's software, and the dissociation constants (Kd values) of BRB and MDM2 proteins were calculated.

[0113] The MST experimental results are as follows Figure 8 As shown, Figure 8 The figure shows the results of a micro-thermophoresis experiment on BRB and MDM2; the horizontal axis BRB Concentration (μM) represents the BRB concentration (μM), and the vertical axis Ratio represents the fluorescence intensity.

[0114] according to Figure 8 It can be seen that the K values ​​of BRB and MDM2 were measured through the MST experiment. D The dissociation constant (dissociation constant) is 8.13 ± 3.05 μM.

[0115] Based on the above analysis, it can be concluded that BRB and MDM2 can bind directly.

[0116] 2.3 Surface Plasmon Resonance Experiment

[0117] Surface plasmon resonance (SPR) experiments were conducted on BRB and MDM2; the experimental procedure was as follows:

[0118] Step 1: After activating the CM5 chip with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, GE Healthcare) and N-hydroxysuccinimide (NHS, GE Healthcare), 20 μg / mL MDM2 protein was fixed in sodium acetate buffer at a flow rate of 10 μL / min for 1200 seconds.

[0119] Step 2, prepare 1×PBS-P containing 5% DMSO. + Run the buffer solution and eliminate refractive index deviation using an 8-point solvent correction curve;

[0120] Step 3: Dilute BRB 2-fold with running buffer (concentration range 1000 μM to 0.49 μM, a total of 12 concentration points), and inject the samples in order from low to high concentration;

[0121] Step 4: Monitor the real-time sensing curves for binding for 60 seconds and dissociation for 90 seconds at a flow rate of 30 μL / min;

[0122] Step 5: After subtracting the reference channel signal using the BIAcoreT200 evaluation software, globally fit a 1:1 Langmuir binding model to obtain the binding and dissociation constants;

[0123] SPR experimental results are as follows Figure 9 As shown; Figure 9 Figure 1 shows the results of surface plasmon resonance experiments on BRB and MDM2.

[0124] according to Figure 9 It can be seen that the KD (Dissociation Constant) value of BRB and MDM2 measured by SPR experiment is 3.263 μM;

[0125] In summary, we can conclude that BRB has a sub-micromolar binding affinity to the MDM2 protein, and its dissociation constant indicates that BRB can form a stable complex. The measured values ​​are ideal, confirming the development potential of BRB as an MDM2 targeting inhibitor.

[0126] 2.3 Validation experiment on the direct effect of BRB on p21 and p53 expression levels

[0127] 2.3.1: Detection of gene expression using real-time quantitative PCR (qRT-PCR)

[0128] Step 1: Genomic DNA Clearance

[0129] Take a 200 μL centrifuge tube, add 1 μg RNA and 4 μL of 4×g DNA Wiper Mix, and add nuclease-free ddH2O to a final volume of 16 μL. Vortex the centrifuge tube for 10–20 seconds and incubate in a PCR instrument at 42℃±0.5℃ for 2 minutes.

[0130] Step 2: Reverse transcription reaction:

[0131] Add 4 μL of 5×HiScriptⅢqRT SuperMix to the reaction solution obtained in step 1 and vortex to mix. Then place it in a PCR instrument for reverse transcription. The PCR program for the reverse transcription reaction is as follows: ① react at 37℃±0.5℃ for 15 minutes; ② denature at 85℃±0.5℃ for 5 seconds; ③ store at 4℃. The obtained cDNA is stored at -20℃.

[0132] Step 3: Preparation of qRT-PCR reaction system:

[0133] Take a 384-well plate and prepare the reaction system according to the following composition (total volume 10 μL / well):

[0134] ① 2 μL of cDNA obtained in step 2

[0135] ② Primer mixture 1 μL

[0136] ③SYBRGreen 5μL

[0137] ④ Nuclease-free ddH2O 2μL

[0138] Step 4: Sample addition and amplification procedure:

[0139] In a 384-well plate, add the reaction mixture prepared in step 3 (cDNA first, then other components) to each well, then seal with an optical sealing membrane and centrifuge to mix. Then, place the 384-well plate into a real-time quantitative PCR instrument for amplification, as shown in the table below:

[0140] Table 1. Amplification Procedure Steps

[0141]

[0142] β-actin was used as an internal control. The relative expression level of each gene was calculated using the 2^(-ΔΔCT) method. The primer sequences used in this experiment are shown in the table below:

[0143] Table 2 shows the gene amplification primer sequences involved in this experiment.

[0144]

[0145] Experimental results are as follows Figure 10 , Figure 11 As shown, Figure 10 A bar chart showing the mRNA levels of P53 in two types of lung cancer cells; Figure 11 A bar chart showing the mRNA levels of P21 in two types of lung cancer cells;

[0146] according to Figure 10 and Figure 11 It can be seen that after BRB treatment, the mRNA expression level of P53 did not change significantly (the change was not statistically significant), while the mRNA expression level of P21 changed significantly.

[0147] Based on the above analysis, it can be concluded that no p53 protein accumulation occurred at the molecular transcriptional level after BRB treatment. Therefore, it is reasonable to speculate that the regulatory effect of BRB on p53 protein accumulation may occur post-transcriptionally.

[0148] 2.3.2 Western Blot Experiment

[0149] Step 1 Sample Preparation

[0150] Take sample cells, perform specific treatment (unless otherwise specified, no specific treatment is required), extract the target protein using a special lysis buffer, and add buffer to prepare the target protein solution sample.

[0151] Step 2: SDS-PAGE electrophoresis

[0152] Determine the target protein concentration obtained in step 1. Based on the target protein concentration determination data, select an appropriate concentration of SDS-PAGE gel rapid preparation kit (purchased from Shanghai Yamei Company) to prepare the gel. Refer to the product instructions for the specific gel preparation process.

[0153] After the gel solidifies, remove the comb vertically to create sample wells. Add a sample containing 30 μg of the target protein to the individual sample wells of the gel, and add a pre-stained protein marker of equal volume to the target protein to the outermost well.

[0154] SDS-PAGE electrophoresis was performed using a two-stage constant voltage mode: 80V was applied during the stacking gel stage, and 120V was applied during the separating gel stage until the indicator migrated to the detection position at the bottom of the gel.

[0155] Step 3: Wet transfer

[0156] Prepare the transfer buffer in advance: Mix 10× Transfer Buffer, methanol, and deionized water in a volume ratio of 1:2:7 and pre-cool on ice. After electrophoresis in step 2, remove the gel and assemble the transfer clip into a "sandwich" structure in sequence: place the pad, filter paper, gel, NC membrane, filter paper, and pad in sequence from the negative electrode to the positive electrode. The entire assembly process should be immersed in the transfer buffer and air bubbles should be removed. Place the prepared transfer clip in the transfer tank, add sufficient pre-cooled transfer buffer, and transfer at a constant current of 350mA for 60–90 minutes under ice bath conditions.

[0157] Step 4: Immunological testing

[0158] Immerse the NC membrane after transfer in step 3 into TBST blocking buffer containing 5% skim milk powder and block at room temperature for 60 minutes. After washing three times with TBST (5 minutes each time), incubate with primary antibody according to the kit instructions, and then incubate at 4°C for 12 hours. The next day, recover and store the primary antibody, and wash the transferred NC membrane three times with TBST (5 minutes each time).

[0159] Step 5: Chemiluminescence detection

[0160] A special developer was selected for development, and a chemiluminescence imager was used to acquire signals and analyze the gray values ​​of the bands.

[0161] Western blotting experiments were performed on H460 cells using steps 1-5 as described above, where step 1 involved:

[0162] The sample cells were selected from H460 cells;

[0163] The specific treatment involved setting up a BRB-treated group and a control group (without BRB treatment) for the sample cells. The BRB-treated group consisted of sample cells first treated with 20 μM BRB. Then, both the BRB-treated group and the control group were treated with CHX for different durations (0.25 hours, 0.5 hours, 1 hour, 2 hours, and 4 hours, respectively), and a blank control group without CHX treatment was also included. "BRB-treated" means that sample cells were taken, added to a culture medium containing a specific concentration of BRB, and co-incubated under suitable conditions. "CHX-treated" means that sample cells were taken, added to a culture medium containing a specific concentration of CHX, and co-incubated under suitable conditions. Unless otherwise specified, "treated with BRB (or other reagents)" in the following text has a similar meaning.

[0164] Experimental results are as follows Figure 12 , Figure 13 As shown;

[0165] Figure 12 The image shows the results of a Western blot experiment on p53 in H460 cells; based on Figure 12 It can be seen that BRB treatment can significantly delay the degradation of p53 protein after CHX treatment and prolong its half-life.

[0166] Figure 13 This is a line graph showing the change in p53 protein levels in H460 cells with different CHX treatment times, where the vertical axis represents the relative expression level of p53 protein; according to Figure 13 As shown, BRB treatment significantly delayed the degradation of p53 protein after CHX treatment and prolonged its half-life.

[0167] Western blotting experiments were performed on A549 cells using steps 1-5 as described above, wherein in step 1:

[0168] The sample cells were selected from A549 cells;

[0169] The specific treatment was as follows: the sample cells were divided into a BRB treatment group and a control group (without BRB treatment). The BRB treatment group was formed by first treating the sample cells with 20 μM BRB. Then, the BRB treatment group and the control group were treated with CHX for different times (0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, respectively) and a blank control group without CHX treatment was set up.

[0170] Experimental results are as follows Figure 14 , Figure 15 As shown.

[0171] Figure 14 The image shows the results of a Western blot experiment on p53 in A549 cells; based on Figure 14 It can be seen that BRB treatment can significantly delay the degradation of p53 protein after CHX treatment and prolong its half-life.

[0172] Figure 15 This is a line graph showing the change in p53 protein levels in A549 cells with different CHX treatment times, where the vertical axis represents the relative expression level of p53 protein; according to Figure 15 It can be seen that BRB treatment can significantly delay the degradation of p53 protein after CHX treatment and prolong its half-life.

[0173] Based on the above experiments and analyses, it can be concluded that BRB treatment can significantly slow down the degradation rate of p53 protein and prolong its half-life.

[0174] 2.3.3 Co-Immunoprecipitation Experiment

[0175] A549 cells and H460 cells were treated with 20 μM BRB, respectively, followed by Co-IP and Western blotting experiments. Co-IP stands for Co-Immunoprecipitation. The specific experimental steps are as follows:

[0176] Step 1: Preparation of cell lysates

[0177] BRB-treated P21 knockout A549 cells were seeded into 10 cm culture dishes and rinsed once with pre-cooled PBS. 1 mL of modified RIPA lysis buffer (containing a mixture of protease inhibitors) was added to each dish, and the cells were lysed on a horizontal shaker at 4 °C for 30 minutes.

[0178] Step 2: Clarification of the pyrolyte

[0179] Collect the lysis buffer into a 1.5 mL centrifuge tube, centrifuge at 4°C (13,000 rpm) for 15 minutes, and then aliquot the supernatant:

[0180] Step 3: Verification of Target Protein Expression

[0181] Take 100 μL of the supernatant obtained in step 2, add an appropriate amount of 5×SDS-PAGE Loading Buffer, and denature in a boiling water bath for 5 minutes (100±2℃). Then, take the target protein for a Western Blot experiment (using the protocol as described in steps 1-5 of section 2.3.2 Western Blot experiment) to detect the expression of the target protein in the host cells. When the expression results are satisfactory, perform an immunoprecipitation experiment.

[0182] Step 4: Beads Preprocessing

[0183] Take the bead stock solution (magnetic bead suspension) and vortex for 30 seconds to fully resuspend it. Using a micropipette, transfer 50 μL of the magnetic bead suspension to a 1.5 mL centrifuge tube, add 1 mL of pre-chilled IP lysis buffer (without protease inhibitors), and vortex for 10 seconds. Centrifuge at 4°C (2,000 rpm) for 3 minutes and discard the supernatant. Repeat the above washing procedure a total of 3 times. After the final centrifugation, completely discard the supernatant, add a small amount of pre-chilled IP lysis buffer, and vortex to resuspend the bead suspension to form a 50% (v / v) working suspension. Store at 4°C for later use.

[0184] Experimental results are as follows Figure 16 , Figure 17 As shown, Figure 16 The image shows the detection results of ubiquitination modification levels in two NSCLCs (A549 and H460). Figure 17 The images show the Co-IP and Western Blot results for two types of NSCLC (A549 and H460).

[0185] according to Figure 16 It can be seen that, using Co-IP and Western Blot analysis with a specific antibody against p53, BRB treatment significantly inhibited the ubiquitination level of p53 protein; according to Figure 17 It can be seen that BRB treatment can inhibit the interaction between endogenous p53 and MDM2.

[0186] Based on the above analysis, it can be concluded that BRB inhibits the ubiquitination and degradation of p53 by affecting the interaction between MDM2 and p53.

[0187] 3. Introduction and experimental verification of the mechanism by which BRB binds to MDM2 to induce senescence in lung cancer cells.

[0188] The aforementioned experiments demonstrating the targeted binding of BRB to MDM2 confirm that BRB and MDM2 bind together. The following section will investigate and experimentally validate the mechanism by which BRB-targeted binding to MDM2 further induces senescence in lung cancer cells:

[0189] Mechanism Introduction: BRB can target and bind to MDM2, and this targeting and binding competes with the binding of p53 to MDM2. Therefore, BRB can induce lung cancer cell senescence by activating the p53 / p21 pathway. Specifically, after competitively binding to MDM2, BRB can block the interaction between MDM2 and p53—that is, BRB can effectively delay the ubiquitination and degradation of p53 by MDM2—thereby inducing lung cancer cell senescence. It should be noted that existing research shows that p53 is a direct upstream transcriptional activator of p21.

[0190] The aforementioned mechanism will be verified through experiments in the following section.

[0191] 3.1 Experiment on BRB-induced senescence in lung cancer cells via the p53 / p21 pathway

[0192] BRB and A549 cells purchased from the market were used. A549 cells were treated with CRISPR / Cas9 technology to knock out P21, resulting in P21-knockout A549 cells. A549 cells were then treated with CRISPR / Cas9 technology to knock out P53, resulting in P53-knockout A549 cells. BRB was then used to treat the P21-knockout A549 cells, resulting in BRB-treated P21-knockout A549 cells. Finally, BRB was used to treat the P53-knockout A549 cells, resulting in BRB-treated P53-knockout A549 cells.

[0193] Western blotting experiments were performed on A549 cells after P21 knockout and A549 cells after P53 knockout (using the same procedure as described in 2.3.2 Western Blot Experiment, the difference being the sample used; the same applies hereinafter) to verify the knockout effect, and the results were as follows. Figure 18 , Figure 19 The experimental results are shown. Figure 18 Figure of Western Blot results for knocking out the P21 gene in A549 cells; Figure 19 The results of the Western Blot experiment of knocking out the P53 gene in A549 cells are shown in the figure; WT represents A549 cells without P21 knockout; P21KO#1 and P21KO#2 are randomly selected A549 cells after P21 knockout; P53KO represents randomly selected A549 cells after P53 knockout.

[0194] from Figure 18 It can be seen that, compared with the wild type, the expression of p21 protein in A549 cells with the p21 gene knocked out was significantly reduced with and without the addition of BRB, indicating that the gene knockout was thorough and effective.

[0195] from Figure 19It can be seen that compared with A549 cells without p53 gene knockout, the expression of p53 protein in A549 cells with p53 gene knockout is significantly reduced, indicating that gene knockout is thorough and effective.

[0196] 3.1.1 SA-β-gal staining experiment (one of the methods)

[0197] Step 1: Cell Treatment

[0198] The A549 cells with P21 knocked out were seeded in 12-well cell culture plates at a density of 1.5 × 10⁻⁶. 4 Cells were cultured per well at 37°C and 5% CO2 using a dedicated culture medium until the logarithmic growth phase. After treatment with 10 μM BRB compound, the culture medium was aspirated, and the cells were washed twice with pre-warmed PBS buffer.

[0199] Step 2: Cell Fixation

[0200] Add 500 μL of β-galactosidase fixative (i.e. β-gal staining fixative) to each well of the 12-well cell culture plate and fix at room temperature (25±1℃) for 15 minutes.

[0201] Step 3 Washing

[0202] Discard the fixative and wash three times with PBS buffer for three minutes each time.

[0203] Step 4: Preparation of staining working solution

[0204] Prepare the staining working solution according to the single-well dosage: 5 μL β-galactosidase staining solution A; 5 μL staining solution B; 465 μL staining solution C; 25 μL X-Gal solution. Mix well and use immediately.

[0205] Step 5: Staining reaction

[0206] Add 500 μL of staining working solution to each well of the 12-well cell culture plate and seal the edges of the plate with sterile sealing film. Incubate in a CO2-free incubator at 37°C in the dark for 12 hours.

[0207] Step 6: Data Collection and Analysis

[0208] Discard the staining solution and wash twice with PBS. Collect images from five randomly selected non-overlapping fields of view under a microscope, and use software to quantify the percentage of cells stained with blue.

[0209] Using the methods described in steps 1 to 6, A549 cells without P21 knockout were re-performed with SA-β-gal staining.

[0210] Using the methods described in steps 1 to 6, A549 cells after P21 knockout were re-treated with SA-β-gal staining. The difference was that BRB was not used to treat the cells in step 1, and only an equal amount of DMSO was added as a control group.

[0211] Using the methods described in steps 1 to 6, A549 cells without P21 knockout were re-treated with SA-β-gal staining. The difference was that BRB was not used to treat the cells in step 1, and only an equal amount of DMSO was added as a control group.

[0212] The results of the aforementioned SA-β-gal staining experiment are as follows Figure 20 , Figure 21 As shown, Figure 20 This is a result of one of the SA-β-gal staining experiments; where "+" indicates cells treated with BRB, and "-" indicates cells not treated with BRB; WT represents A549 cells without P21 knockout; P21KO#1 and P21KO#2 are randomly selected A549 cells after P21 knockout. Figure 21 A bar chart showing the percentage of senescent A549 cells in the treatment with / without BRB addition and the treatment with / without P21 gene knockout; the vertical axis “Sensitive percentage (%)” represents the percentage of senescent cells in the total number of cells.

[0213] from Figure 20 It can be seen that knocking out P21 can partially reduce the number of BRB-induced SA-β-gal staining positive cells, and the difference is statistically significant.

[0214] from Figure 21 It can be seen that knocking out P21 can partially reduce the number of BRB-induced SA-β-gal positive cells, and the difference is statistically significant.

[0215] Based on the above analysis, it can be concluded that BRB induces p21-dependent senescence in lung cancer cells.

[0216] 3.1.2 SA-β-gal staining experiment part two

[0217] SA-β-gal staining experiment was performed on the A549 cells after P53 knockout. The procedure of the SA-β-gal staining experiment is the same as one of the SA-β-gal staining experiments in 3.1.1 above.

[0218] Using the above experimental method, A549 cells without P53 knockout were re-performed with SA-β-gal staining.

[0219] Using the above experimental method, A549 cells with P53 knocked out were re-stained with SA-β-gal, except that BRB was not used to treat the cells, and only an equal amount of DMSO was added as a control group.

[0220] Using the above experimental method, A549 cells without P53 knockout were re-stained with SA-β-gal, except that BRB was not used to treat the cells, and only an equal amount of DMSO was added as a control group.

[0221] The results of the aforementioned SA-β-gal staining experiment are as follows Figure 22 , Figure 23 As shown, Figure 22 The image shows the results of the SA-β-gal staining experiment; "+" indicates cells treated with BRB, "-" indicates cells not treated with BRB; WT represents A549 cells without P53 knockout; P53KO represents randomly selected A549 cells with P53 knockout.

[0222] Figure 23 A bar chart showing the percentage of senescent A549 cells in the treatment with / without BRB addition and the treatment with / without P53 gene knockout; the vertical axis “Sensitive Percentage (%)” represents the percentage of senescent cells in the total number of cells, and the same applies below.

[0223] from Figure 22 , Figure 23 It can be seen that knocking out P53 can partially reduce the number of BRB-induced SA-β-gal positive cells, and the difference is statistically significant.

[0224] Based on the above analysis, it can be concluded that BRB induces lung cancer cell senescence through the p53 / p21 pathway.

[0225] 3.2 BRB-induced cellular senescence experiments in A549 and H460 cells

[0226] Existing research indicates that a sustained DNA damage response (DDR) is a marker of cellular senescence. γ-H2AX is frequently used as a biomarker for DDR due to its close relationship with it. Furthermore, γ-H2AX protein expression is upregulated after DNA damage, and nuclear integrity is impaired due to the loss of Lamin B1. Therefore, Lamin B1 is also considered a biomarker of cellular senescence. The following experiments use γ-H2AX and DDR as biomarkers to verify the senescence-inducing effect of BRB on A549 and H460 cells.

[0227] BRB purchased from the market, along with A549 and H460 cells, were used to treat A549 and H460 cells to obtain BRB-treated A549 cells and BRB-treated H460 cells.

[0228] 3.2.1 Immunofluorescence assay

[0229] Immunofluorescence experiments were performed on BRB-treated A549 cells and BRB-treated H460 cells to detect the expression levels of DNA damage markers (53BP1, γ-H2AX). The steps are as follows:

[0230] Step 1: Cell Treatment and Fixation

[0231] A549 and H460 cells were seeded separately in 35 mm confocal culture dishes at a density of 3 × 10⁻⁶ cells / mL. 4 Cells were cultured per dish for 24 hours in a dedicated culture medium to allow them to adhere. Then, the medium was replaced with fresh medium containing 20 μM BRB compound and cultured for another 48 hours. The medium was then discarded, and 1 mL of pre-chilled 4% paraformaldehyde solution was added to each dish to fix the cells.

[0232] Step 2: Cell permeation and blocking

[0233] Remove the fixative and wash three times with PBS buffer. Add PBS solution containing 0.1% Triton X-100 and permeate at room temperature for 10 minutes. After permeation, add PBS solution containing 2% BSA and block at room temperature for 60 minutes.

[0234] Step 3: Primary Antibody Incubation

[0235] Remove the blocking solution and add diluted primary antibody working solution: MDM2 and p53 specific antibody labels from different species (mouse and rabbit, respectively), and incubate at 4°C for 12 hours (overnight).

[0236] Step 4: Secondary antibody labeling and nuclear staining

[0237] Discard the primary antibody and wash three times with PBST buffer (5 minutes each time). Add 200 μL of mixed fluorescent secondary antibody working solution: fluorescent secondary antibodies labeled with different fluorescein, either goat anti-mouse or anti-rabbit (FITC, green fluorescence; Rhodamine, red fluorescence), and incubate at room temperature for 60 minutes in the dark. After washing three times with PBST, add DAPI solution to stain the nuclei in the dark. After staining, photograph and record the results using a fluorescence microscope.

[0238] The immunofluorescence experiment was repeated using the same experimental method, except that BRB was not used to treat the cells.

[0239] The results of the immunofluorescence experiment are as followsFigure 24 , Figure 25 As shown in the figure, Figure 24 Immunofluorescence images of two types of lung cancer cells; from Figure 24 It can be seen that the number of DNA damage foci with high expression of γ-H2AX in A549 cells and H460 cells increased significantly after BRB treatment, and the difference was statistically significant.

[0240] Based on the above analysis, it can be concluded that the number of DNA damage foci with high expression of γ-H2AX in A549 and H460 cells increased significantly after BRB treatment, which indicates that A549 and H460 cells underwent senescence after BRB treatment.

[0241] Figure 25 The percentage of γ-H2AX-positive cells in the two lung cancer cell lines was statistically analyzed by randomly selecting five fields of view; from Figure 25 It can be seen that the number of γ-H2AX positive cells in A549 cells and H460 cells increased significantly after BRB treatment, and the difference was statistically significant.

[0242] Based on the above analysis, it can be concluded that the number of DNA damage foci with high expression of γ-H2AX in A549 and H460 cells increased significantly after BRB treatment, and A549 and H460 cells underwent senescence after BRB treatment.

[0243] 3.2.2 Western Blot Experiment

[0244] Western blotting experiments were performed on BRB-treated A549 cells and BRB-treated H460 cells (using steps 1-5 of section 2.3.2 Western Blot, the difference being the sample cells and specific treatments). The BRB treatment was performed using concentration gradients of 0, 5, 10, and 20 μM. The final Western blotting results are shown below. Figure 26 As shown; Figure 26 Western blot images of Lamin B1 cells from two lung cancer cell lines;

[0245] from Figure 26 It can be seen that the expression of Lamin B1 in A549 cells and H460 cells decreased after BRB treatment;

[0246] Based on the above analysis, it can be concluded that the expression of Lamin B1 in A549 cells and H460 cells decreased after BRB treatment, suggesting that both types of lung cancer cells underwent senescence after BRB treatment.

[0247] 3.3 BRB-induced cellular senescence assay in NSCLC lineage cells

[0248] BRB and NSCLC lineage cells (H1975 cells, A549 cells, H2030 cells, H1650 cells, H460 cells, 1299 cells, and EKVX cells) were used to conduct a BRB-induced cellular senescence assay in NSCLC lineage cells. The experimental procedure was the same as one of the SA-β-gal staining experiments described in 3.1.1 above, with BRB concentration gradients of 0, 5, 10, and 20 μM. In the figure, P53WT indicates that the cells are P53 wild-type cells, P53null indicates that the cells are P53-deficient cells, and P53mut indicates that the cells are P53 mutant cells.

[0249] Experimental results are as follows Figure 27 , Figure 28 As shown, Figure 27 Senescence staining of NSCLC lineage cells after treatment with different concentrations of BRB; Figure 28 A bar chart showing the percentage of senescent cells after treating NSCLC lineages with different concentrations of BRB;

[0250] according to Figure 27 , Figure 28 It can be seen that BRB treatment can induce an increase in the proportion of SA-β-gal positive cells in several P53 wild-type NSCLC cell lines in a concentration-dependent manner, including H1975, A549, H2030, H1650, and H460. However, SA-β-gal staining experiments showed that BRB treatment had no significant effect on P53 mutant or deletion-type NSCLC cell lines, including 1299 cells and EKVX cells.

[0251] Based on the above analysis, it can be concluded that BRB can selectively induce senescence in wild-type P53 NSCLC cells, but has no significant effect on P53 mutant or deleted NSCLC cell lines.

[0252] Example 2: Antitumor efficacy experiment of berberine (animal experiment)

[0253] 1. Experimental Materials

[0254] 1.1 Laboratory animals and their housing environment

[0255] Experimental animals: The animals used in this study were BALB / c-nu mice, BALB / c nude mice, which were free of special pathogens (SPF), female, 5 weeks old, and weighed 20±2g. They were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0256] Husbandry conditions: SPF grade, well-ventilated cages, temperature 20-26℃, 12-hour light and dark cycle per day, ample food and water supply, acclimatization period of one week. Approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM2311130005); the maximum permissible tumor volume is set at 2000 mm². 3 The procurement, feeding, and use of laboratory animals have all been approved by the Animal Experiment Management Committee of Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine.

[0257] 1.2 Experimental reagents

[0258] Test drug: BRB purchased in Example 1 was used as the test drug in this example;

[0259] Control group drug: cisplatin, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., CAS No.: 15663-27-1;

[0260] 2. Experimental Methods

[0261] 2.1 Construction of a mouse model of lung cancer:

[0262] The aforementioned experimental animals (mice) were inoculated with A549 cells, and the model construction steps are as follows:

[0263] (1) Using A549 cells in the logarithmic growth phase, after adding trypsin, digestion was terminated with DMEM complete medium (Dulbecco's Modified Eagle's Medium, hereinafter referred to as DMEM), and the cell suspension was collected into a 15mL centrifuge tube and centrifuged at 1200rpm for 3min.

[0264] (2) Wash with PBS, centrifuge, and discard the supernatant;

[0265] (3) Resuspend cells in PBS, count cells, and adjust cell concentration to 2×10⁻⁶. 6 125 μL of cells were placed on ice.

[0266] (4) The cell suspension was extracted with an insulin needle and 125 μL of A549 cell suspension was injected subcutaneously into the back of each mouse to construct a mouse subcutaneous xenograft model.

[0267] 2.2 Experimental grouping and administration method:

[0268] Forty experimental mice were randomly divided into 5 groups. The grouping and administration methods are described below:

[0269] A549 modeling + low-dose BRB treatment group: Eight A549 modeling experimental mice were used. Twenty-four hours after modeling, mice were intraperitoneally injected with 50 mg / kg of BRB once daily until sacrifice. In the accompanying diagram of the instruction manual, the A549 modeling + low-dose BRB treatment group is indicated as BRB 50 mg / kg or 50 mg / kg.

[0270] A549 modeling + medium-dose BRB treatment group: Eight A549 modeling experimental mice were used. Twenty-four hours after modeling, mice were intraperitoneally injected with 100 mg / kg of BRB once daily until sacrifice. In the accompanying diagram of the instruction manual, the A549 modeling + medium-dose BRB treatment group is indicated as BRB 100 mg / kg or 100 mg / kg.

[0271] A549 modeling + high-dose BRB treatment group: Eight A549 modeling experimental mice were used. Twenty-four hours after modeling, mice were intraperitoneally injected with 150 mg / kg of BRB once daily until sacrifice. In the accompanying diagram of the instruction manual, the A549 modeling + high-dose BRB treatment group is indicated as BRB 150 mg / kg or 150 mg / kg.

[0272] Placebo group: Eight A549 model mice were used. Twenty-four hours after modeling, DMSO was administered intraperitoneally at a dose of 2 mg / kg once daily until the mice were sacrificed. In the accompanying diagram of the product information, the placebo group is indicated as Ctrl or Vehicle.

[0273] Positive control group: Eight A549 model mice were used. Twenty-four hours after modeling, cisplatin (2 mg / kg) was administered intraperitoneally every two days until the mice were sacrificed. The placebo group is represented as CDDP in the accompanying diagram of the product information.

[0274] 2.3 Data Processing

[0275] All experimental results are presented as mean ± standard deviation (mean ± SD). Data analysis was performed using GraphPadPrism version 8. Pairwise comparisons among multiple groups were performed using one-way ANOVA, and comparisons between two groups were performed using two-tailed unpaired t-tests. All tests were presented at four significance levels (P < 0.05 was *, P < 0.01 was **, P < 0.001 was ***, and ns indicated no statistical difference).

[0276] 2.4 Weight Change Detection

[0277] Detection method description: Five days after the mice were modeled, their weight was measured and recorded using an electronic scale to detect their weight status.

[0278] Test samples: All mice;

[0279] Testing frequency: Five days after the mice were modeled, they were weighed for the first time, and then their weight was measured every two days thereafter; the mice were weighed for the last time before they were sacrificed.

[0280] Detection indicator: Mouse body weight.

[0281] The test results obtained after detecting changes in weight are as follows: Figure 29 As shown, from Figure 29 It can be seen that there was no significant change in the body weight of mice after BRB treatment; based on the above analysis, it can be concluded that BRB has no obvious toxic side effects in vivo.

[0282] 2.5 Detection of changes in subcutaneous tumor volume

[0283] Detection method: Five days after mouse modeling, the length and width of the tumor were measured using calipers, and the tumor volume was calculated using the formula V = L × S. 2 / 2 (L is the longest diameter, S is the shortest diameter); Detecting tumor growth in mice;

[0284] Test samples: All mice;

[0285] Testing frequency: The first measurement was performed 5 days after the mice were modeled, followed by measurements every two days thereafter; the last measurement was performed before the mice were sacrificed.

[0286] Detection indicator: tumor volume in mice.

[0287] The results obtained after detecting changes in the volume of subcutaneous tumors are as follows: Figure 30 As shown, from Figure 30 It can be seen that by calculating the volume of subcutaneous tumors, the tumor volume in the A549 modeling group + low / medium / high dose BRB treatment group was smaller than that in the placebo group; based on the above analysis, it can be concluded that BRB treatment significantly inhibited the growth of subcutaneous tumors in mice.

[0288] 2.6 Subcutaneous tumor weighing detection

[0289] Detection method: On day 23 after A549 modeling, mice were sacrificed to obtain subcutaneous xenografts. The subcutaneous xenografts were weighed and photographed to compare the size of the subcutaneous tumor tissue in different treatment groups.

[0290] Test samples: All mice;

[0291] Detection frequency: once, on day 23 of the experiment;

[0292] Detection indicators: weight and morphology of subcutaneous xenografts in mice.

[0293] After euthanizing the mice, the subcutaneous tumors were dissected and removed. The morphology of the subcutaneous transplanted tumors was observed, and the results were as follows: Figure 31 As shown, from Figure 31 As can be seen from the subcutaneous tumor imaging results, the tumor volume in the A549 modeling + low / medium / high dose BRB treatment group was smaller than that in the placebo group. Based on the above analysis, we can conclude that the tumor volume in the A549 modeling + low / medium / high dose BRB treatment group was smaller than that in the placebo group, suggesting that BRB treatment significantly inhibited the growth of subcutaneous tumors in mice.

[0294] After euthanizing the mice, the subcutaneous tumors were dissected and removed. The subcutaneous transplanted tumors were weighed, and the results were as follows: Figure 32 As shown, from Figure 32 It can be seen that, compared with the placebo group, the subcutaneous tumor weight of the A549 modeling group + low / medium / high dose BRB treatment group was smaller than that of the placebo group, and the difference was statistically significant. Based on the above analysis, it can be concluded that BRB treatment significantly inhibits the growth of subcutaneous tumors in mice.

[0295] 2.7 In vivo safety studies of the drug

[0296] Detection method introduction: On day 23 after A549 modeling, mice were sacrificed and dissected to conduct in vivo safety experiments. The experimental method was to perform HE staining on the liver and kidneys, collect mouse serum to detect liver and kidney function indicators (ALT, AST, UN, CREA), and weigh the mice to measure the ratio of liver and kidney weight to body weight.

[0297] Test samples: All mice;

[0298] Detection frequency: once, on day 23 of the experiment;

[0299] Testing indicators: Liver and kidney function indicators (ALT, AST, UN, CREA), etc.

[0300] (1) Hematoxylin-eosin staining, hereinafter referred to as HE staining, is one of the commonly used staining methods in paraffin sectioning. Liver and kidney HE staining experiment: After euthanizing mice, their livers and kidneys are dissected and HE staining is performed to obtain the following results: Figure 33 , Figure 34 The experimental results shown are as follows, in which Figure 33 For the liver, Figure 34 For the kidneys;

[0301] from Figure 33 , Figure 34It can be seen from the HE staining results of the liver and kidneys of mice that there was no significant difference in the liver and kidney tissue structure between the A549 modeling group and the low / medium / high dose BRB treatment group and the placebo group.

[0302] Based on the above analysis, we can conclude that BRB has no obvious toxic side effects on the liver and kidney tissues of mice, suggesting that BRB is safe for in vivo administration.

[0303] (2) Detection of liver and kidney function indicators (ALT, AST, UN, CREA): After euthanizing the mice, serum was collected for the detection of liver and kidney function indicators (ALT, AST, UN, CREA), as shown below. Figure 35 , Figure 36 , Figure 37 , Figure 38 The experimental results are shown below;

[0304] ALT stands for alanine aminotransferase, hereinafter referred to as ALT; AST stands for aspartate aminotransferase, hereinafter referred to as AST; UN stands for urea nitrogen, hereinafter referred to as UN; and CREA stands for creatinine, hereinafter referred to as CREA.

[0305] from Figure 35 , Figure 36 , Figure 37 , Figure 38 It can be seen that the results of liver and kidney function indicators in mice indicate that there is no significant difference in liver and kidney tissue function between mice in the A549 modeling + low / medium / high dose BRB treatment group and the placebo group.

[0306] Based on the above analysis, we can conclude that BRB has no obvious toxic side effects on the liver and kidney function of mice, suggesting that BRB is safe for in vivo use.

[0307] (3) Mouse liver and kidney weight to body weight ratio test: After euthanizing the mice, their livers and kidneys were dissected and weighed. The ratio of liver and kidney weight to body weight was calculated, as shown in the figure. Figure 39 , Figure 40 The experimental results are shown below;

[0308] from Figure 39 , Figure 40 It can be seen that the results of liver weight / body weight ratio and kidney weight / body weight ratio in mice indicate that there is no significant difference in liver weight / body weight ratio and kidney weight / body weight ratio between mice in the A549 modeling + low / medium / high dose BRB treatment group and the placebo group.

[0309] Based on the above analysis, we can conclude that BRB has no obvious toxic side effects on the liver and kidney tissues of mice, suggesting that BRB is safe for in vivo administration.

Claims

1. The application of berberine in the preparation of drugs for the prevention and treatment of lung cancer, characterized in that, The lung cancer in question is non-small cell lung cancer that expresses wild-type p53 protein.

2. The application according to claim 1, characterized in that, The drug induces tumor cell senescence by inhibiting the protein interaction between MDM2 and p53, thereby prolonging the half-life of the p53 protein.

3. The application according to claim 1, characterized in that, The drug inhibited the growth of A549 subcutaneous xenografts in animal models.

4. The application according to any one of claims 1 to 3, characterized in that, The lung cancer drug is a pharmaceutical composition comprising: a. Berberine or its pharmaceutically acceptable salt; b. A pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The carrier includes at least one of microspheres, liposomes, nanoparticles, and microemulsions.

6. The application of berberine in the preparation of MDM2 inhibitors, characterized in that, In non-small cell lung cancer expressing wild-type p53 protein, the MDM2 inhibitor can specifically bind to the MDM2 protein, inhibit p53 ubiquitination and degradation, and upregulate p21 mRNA expression.

7. The application according to any one of claims 6, characterized in that, The MDM2 inhibitor is a pharmaceutical composition comprising: a. Berberine or its pharmaceutically acceptable salt; b. A pharmaceutically acceptable carrier.

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