Application of semen ziziphi spinosae in preparation of anti-hepatoma medicine and action mechanism analysis method

Abstract

The invention belongs to the field of pharmacological mechanism research, and particularly relates to application of spina date seeds in preparation of anti-hepatoma drugs and an action mechanism analysis method. The active component of the spina date seed in preparation of the anti-hepatoma drug is spina date seed saponin B. According to the step analysis of spina date seed active component screening and target spot prediction, hepatoma target spot screening, pathway enrichment analysis, PPI network analysis, molecular docking and biological verification, the application of spina date seed in preparation of the anti-hepatoma drug is known. The spina date seed active substance can play a role in liver cancer through a PI3K-AKT signal channel, and a direction is pointed out for subsequent development and application of anti-liver cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmacological mechanism research, specifically relating to the application of jujube seed in the preparation of anti-liver cancer drugs and the method for analyzing its mechanism of action. Background Technology

[0002] Liver cancer is one of the most prevalent malignant tumors worldwide, with both high incidence and mortality rates. According to the World Health Organization, more than 900,000 new cases of liver cancer are diagnosed globally each year, with approximately 830,000 deaths, and the incidence rate is increasing annually. Liver cancer is characterized by its insidious onset, rapid progression, difficulty in early diagnosis, and poor prognosis; most patients are diagnosed at an advanced stage, missing the opportunity for radical surgical treatment. Currently, commonly used clinical treatments include surgical resection, liver transplantation, interventional therapy, targeted drugs (such as sorafenib and lenvatinib), and immunotherapy, but these methods suffer from limited efficacy, frequent drug resistance, and significant side effects. Therefore, developing highly effective, low-toxicity anti-liver cancer drugs and treatment strategies with clearly defined mechanisms of action is an urgent need in the field of oncology research.

[0003] Some extracts or active ingredients of jujube seed have inhibitory effects on various tumor cells. For example, total saponins of jujube seed can induce tumor cell apoptosis and inhibit cell proliferation and migration, exhibiting anti-tumor activity in models of lung cancer and breast cancer; its flavonoid components can affect the tumor microenvironment by regulating oxidative stress and inflammatory responses. However, systematic research on jujube seed and its active ingredients in the field of anti-liver cancer is still relatively scarce. The specific targets, key regulatory pathways, and molecular mechanisms have not yet been clarified, limiting its development and application as an anti-liver cancer drug. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an application and mechanism analysis method for jujube seed in the preparation of anti-liver cancer drugs, identifies the effective components of jujube seed in the preparation of anti-liver cancer drugs, and explains its mechanism of action.

[0005] The specific technical solution adopted in this invention is as follows:

[0006] The application of jujube seed in the preparation of anti-liver cancer drugs, wherein the active ingredient of jujube seed is jujube seed saponin B.

[0007] A method for analyzing the mechanism of action of jujube seed in the preparation of anti-liver cancer drugs, characterized by the following steps:

[0008] S1. Screening and target prediction of active ingredients in jujube seed:

[0009] Information on bioactive components in jujube seed was obtained by screening the TCMSP database, and then the chemical formulas and PubChem_CIDs of the above bioactive components in jujube seed were obtained by using the PubChem database.

[0010] Using the SwissTargetPrediction database, potential targets of action were identified based on the chemical formulas of the canonical smiles of bioactive components, with humans selected as the species. Simultaneously, the targets were screened using the BATMAN-TCM database based on Pubchem_CID. The bioactive components and targets obtained from the two databases were combined to obtain the targets of action of the bioactive components of jujube seed.

[0011] S2. Liver cancer target screening:

[0012] Using the GeneCards database, the keyword "hepatocellular carcinoma" was entered to retrieve liver cancer targets related to liver cancer. The intersection of liver cancer targets and the targets of active components of jujube seed was obtained to obtain the anti-liver cancer targets of active components of jujube seed.

[0013] S3. Pathway enrichment analysis:

[0014] The active ingredients of Ziziphus jujuba seed were input into the DAVID database for GO and KEGG pathway enrichment analysis, and the PI3K-AKT signaling pathway was significantly enriched.

[0015] S4.PPI Network Analysis:

[0016] The active ingredients of Ziziphus jujuba seed were input into the STRING database to obtain the interaction information between the active ingredients of Ziziphus jujuba seed and the anti-liver cancer target; the Cytohubba plugin in Cytoscape software was used to perform molecular analysis of key targets to obtain the key target molecule AKT1.

[0017] S5. Molecular docking:

[0018] Molecular docking was performed between the bioactive components information of jujube seed and key target molecules, revealing that the interaction between jujube seed saponin B and the key target molecule AKT1 is closely related to liver cancer.

[0019] S6. Biological verification: It was confirmed that jujube seed saponin B has an anti-liver cancer mechanism.

[0020] In step S1, 33 active ingredients of jujube seed were screened using TCMSP; the canonical samples of bioactive substances were searched using the Pubchem database, and then the targets of the active substances were searched using the SwissTargetPrediction database. After removing duplicate targets, 28 active substances and 751 targets were obtained; the Pubchem CID of bioactive substances was searched using the Pubchem database, and then the targets of the active substances were searched using the BATMAN-TCM database, resulting in 27 active ingredients and 580 targets; finally, the intersection of the targets obtained from the two databases yielded 26 bioactive ingredients of jujube seed and 144 corresponding targets.

[0021] In step S2, a set of liver cancer-related genes was screened through the GeneCards database. Based on the keyword "hepatocellular carcinoma", a total of 8396 genes were obtained. The intersection of 144 target molecules with the 8396 liver cancer target molecules yielded 105 identical genes, which are the anti-liver cancer target molecules of jujube seed active ingredients.

[0022] In step S3, the 105 active ingredients of jujube seed obtained from the above analysis were subjected to GO and KEGG enrichment analysis using the DAVID database. The corrected p < 0.05 was used as the screening condition in the results.

[0023] In step S6, the biological verification includes cell experiment verification and animal experiment verification.

[0024] The beneficial effects of this invention are:

[0025] This invention proposes that the active ingredient in the preparation of anti-liver cancer drugs from jujube seed is jujube seed saponin B. Furthermore, through the analysis of the mechanism of action of the active ingredient in this invention, it is known that the active substances in jujube seed can exert their effects on liver cancer through the PI3K-AKT signaling pathway, thus pointing the way for the development and application of subsequent anti-liver cancer drugs. Attached Figure Description

[0026] Figure 1 Venn diagram screening for active ingredients in jujube seed;

[0027] Figure 2 Venn diagram of the target genes of active substances in jujube seed and liver cancer target genes;

[0028] Figure 3 GO enrichment analysis;

[0029] Figure 4 KEGG enrichment analysis;

[0030] Figure 5 KEGG enrichment pathway analysis;

[0031] Figure 6 The effect of JUB on the proliferation of liver cancer cells and normal liver cells;

[0032] A: Effects of different concentrations of JUB and different intervention times on the proliferation of HepG2 liver cancer cells.

[0033] B: Effects of different concentrations of JUB and different intervention times on the proliferation of normal LO2 liver cells.

[0034] *: At the same treatment time, p<0.05 compared with the control group;

[0035] ***: At the same time point, compared with the control group, p<0.001; n≥3.

[0036] Figure 7 JUB induces apoptosis in liver cancer cells;

[0037] A: The effect of different concentrations of JUB intervention on apoptosis in HepG2 liver cancer cells.

[0038] B: Statistics on apoptosis.

[0039] C: Effects of different concentrations of JUB intervention on apoptosis in normal LO2 liver cells.

[0040] D: Statistics on apoptosis.

[0041] *: At the same treatment time, p<0.05 compared with the control group. n≥3.

[0042] Figure 8 The relative expression of BAX and BCL-2 proteins after JUB intervention in HepG2 cells;

[0043] * indicates a significant difference compared to the control group. (P < 0.05)

[0044] Figure 9 JUB inhibits the migration of liver cancer cells;

[0045] A: The effect of different concentrations of JUB intervention on the migration of HepG2 liver cancer cells.

[0046] B: Statistical chart of cell migration.

[0047] *: At the same treatment time, p<0.05 compared with the control group;

[0048] **: At the same treatment time, p<0.01 compared with the control group;

[0049] ***: At the same treatment time, p<0.001 compared with the control group, n=3.

[0050] Figure 10 The relative expression of PI3K and p-PI3K proteins after JUB intervention in HepG2 cells;

[0051] * indicates a significant difference compared to the control group. (P < 0.05)

[0052] # indicates a significant difference compared to the JUB (120 μM) intervention group. (P < 0.05)

[0053] Figure 11 The relative expression of Akt and p-Akt proteins after JUB intervention in HepG2 cells;

[0054] * indicates a significant difference compared to the control group. (P < 0.05)

[0055] # indicates a significant difference compared to the JUB (120 μM) intervention group. (P < 0.05)

[0056] Figure 12 Photographs and HE-stained sections of liver tissue during the DEN-induced hepatocellular carcinoma model in SD rats;

[0057] Figure 13 Images of the livers of SD rats in each experimental group;

[0058] Figure 14 HE staining images of SD rat livers from each experimental group;

[0059] Figure 15 DEN-induced liver cancer model in SD rats and changes in rat body weight during intervention;

[0060] * indicates a significant difference between the DEN group and the control group. (P < 0.05)

[0061] # indicates a significant difference between the DEN+JUB (40μM) group and the control group. (P<0.05)

[0062] △ indicates a significant difference between the DEN+JUB (80μM) group and the control group. (P<0.05) Figure 16 DEN-induced liver cancer model in SD rats and comparison of food intake during intervention;

[0063] * indicates a significant difference compared to the control group. (P < 0.05)

[0064] # indicates a significant difference compared to DEN+JUB (120 μM). (P < 0.05)

[0065] △ indicates a significant difference compared to the DEN+JUB (80 μM) group. (P < 0.05)

[0066] Figure 17 Comparison chart of rat liver indices;

[0067] * indicates a significant difference compared to the control group. (P < 0.05)

[0068] Figure 18 Immunohistochemical images and quantitative analysis of PI3K protein in rat liver tissue;

[0069] Figure 19 Immunohistochemical images and quantitative analysis of Akt protein in rat liver tissue;

[0070] * indicates a significant difference compared to the control group. (P < 0.05)

[0071] Figure 20 Immunohistochemical images and quantitative analysis of p-PI3K protein in rat liver tissue;

[0072] * indicates a significant difference compared to the control group. (P < 0.05)

[0073] # indicates a significant difference compared to the DEN group. (P < 0.05)

[0074] △ indicates a significant difference compared to the DEN+JUB (40 μM) group. (P < 0.05)

[0075] Figure 21 Immunohistochemical images and quantitative analysis of p-Akt protein in rat liver tissue;

[0076] * indicates a significant difference compared to the control group. (P < 0.05)

[0077] # indicates a significant difference compared to the DEN group. (P < 0.05)

[0078] △ indicates a significant difference compared to the DEN+JUB (40 μM) group. (P < 0.05)

[0079] Figure 22 Immunohistochemical images and quantitative analysis of BAX protein in rat liver tissue;

[0080] * indicates a significant difference compared to the control group. (P < 0.05)

[0081] # indicates a significant difference compared to the DEN group. (P < 0.05)

[0082] Figure 23 Immunohistochemical images and quantitative analysis of BCL-2 protein in rat liver tissue;

[0083] * indicates a significant difference compared to the control group. (P < 0.05)

[0084] # indicates a significant difference compared to the DEN group. (P < 0.05)

[0085] Figure 24 Docking diagram of JUB with the key protein molecule AKT1; Detailed Implementation

[0086] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0087] In a specific embodiment, the present invention discloses an application and mechanism of action analysis of jujube seed in the preparation of anti-liver cancer drugs, which specifically includes the following steps:

[0088] 1. Screening and target prediction of active ingredients in jujube seed

[0089] Thirty-three active components of jujube seed were identified using TCMSP screening. Canonical samples of the bioactive substances were searched in the Pubchem database, and then targets were identified using the SwissTargetPrediction database. After removing duplicate targets, 28 active substances and 751 targets were obtained. Pubchem CIDs of the bioactive substances were searched in the Pubchem database, and then targets were identified using the BATMAN-TCM database, resulting in 27 active components and 580 targets. Finally, the intersection of the targets obtained from the two databases yielded 26 bioactive components of jujube seed and 144 corresponding targets. See Table 1 (Bioactive Components and Targets of Jujube Seed) for details. Figure 1 .

[0090] Table 1 Bioactive components and targets of jujube seed

[0091]

[0092]

[0093]

[0094]

[0095] 2. Screening of target genes for liver cancer

[0096] A gene set related to liver cancer was screened using the GeneCards database. A search based on the keyword "hepatocellular carcinoma" yielded 8396 genes.

[0097] 3. Screening of anti-hepatocellular carcinoma targets of active ingredients in jujube seed

[0098] Taking the intersection of the 144 target molecules of the bioactive components of jujube seed obtained above with 8396 target molecules of liver cancer, a total of 105 identical genes were obtained, which are the anti-liver cancer targets of the bioactive components of jujube seed. The results are as follows. Figure 2 As shown.

[0099] 4. Enrichment analysis of GO and KEGG pathways

[0100] GO and KEGG enrichment analyses were performed on the 105 active ingredients of Ziziphus jujuba seed identified in the above analysis using the DAVID database. The results were selected with a corrected p < 0.05 as the screening criterion. GO analysis categorized gene functions into three types: biological process (BP), cellular component (CC), and molecular function (MF). Based on the number of molecules enriched in each pathway, the top 10 pathways with the highest enrichment were selected for presentation. The results are shown below. Figure 3 As shown in the figure. Twenty pathways significantly enriched in KEGG are displayed, and the results are as follows. Figure 4 As shown in the figure, the above results indicate that the PI3K-AKT signaling pathway was significantly enriched, suggesting that the active substances in jujube seed can exert their effects on liver cancer through the PI3K-AKT signaling pathway.

[0101] 5. Protein-protein interaction analysis

[0102] The active ingredients of Ziziphus jujuba seed targeting liver cancer were input into the SRTING database to obtain information on the interactions between targets. Using the Cytohubba plugin in Cytoscape, the top 50 molecules were obtained according to their degree, as detailed in Table 2. The top 5 molecules were SRC, AKT1, ESR1, TP53, and EGFR.

[0103] To further screen the anti-liver cancer mechanism of active ingredients in jujube seed, enrichment pathway analysis was performed on the top 50 molecules identified. The results showed that the PI3K / AKT signaling pathway was significantly enriched in the KEGG pathway analysis. Figure 5 The results showed consistency with the enrichment of target points for the anti-liver cancer activity of active ingredients in jujube seed.

[0104] 6. Molecular docking analysis

[0105] Analytical methods: The X-ray crystal structure of AKT (PDB:7NH5) was obtained from a protein database, and the compound was extended to a 3D structure using OpenBabel. The receptor protein and ligand were processed using the AutoDock tool (ADT3), and molecular docking was performed using AutodockVina (1.2.0). Finally, a protein-ligand interaction map was generated using PyMOL. Figure 24 ).

[0106] Molecular docking analysis was performed on jujube seed bioactive substance saponin B and the screened key target AKT1. Analysis of the discovered interaction forces revealed that the receptor protein and the small molecule directly formed multiple sets of interaction forces, such as the hydrogen bond between AKT's GLY394 and the ligand. Under the influence of these interactions, the binding energy of the protein-small molecule complex was -10.2 kcal / mol, exhibiting excellent overall performance, indicating that jujube seed bioactive substance saponin B can bind to AKT1.

[0107] 7. Cellular experimental verification

[0108] 7.1 JUB inhibits the proliferation of liver cancer cells.

[0109] To investigate the effect of JUB on the proliferation of liver cancer cells and the appropriate intervention concentration, the CCK-8 assay kit was used to determine the effect of different concentrations and treatment times of JUB on the proliferation of liver cancer cells. The results are as follows: Figure 6 As shown, 80 μM JUB can inhibit the activity of HepG2 liver cancer cells, but has no effect on normal liver cells LO2. The EC50 of JUB intervention in normal liver cells LO2 for 24 h was 165.2 (156.8-173.5) μM, and for 48 h it was 135.3 (122.7-148) μM. The EC50 of JUB intervention in HepG2 liver cancer cells for 24 h was 109.8 (104.5-115) μM, and for 48 h it was 90.31 (72.85-107.8) μM. Based on the experimental results and previous studies, the final JUB intervention concentrations for subsequent treatment of liver cancer cells were determined to be 40, 80, and 120 μM.

[0110] 7.2 JUB induces apoptosis in liver cancer cells

[0111] Flow cytometry with Annexin V-FITC and PI double staining was used to analyze whether JUB's inhibition of the proliferation of HepG2 liver cancer cells and normal LO2 liver cells was related to apoptosis. Results are as follows: Figure 7 As shown, compared with the control group, JUB intervention promoted apoptosis in liver cancer cells in a dose-dependent manner. However, it did not induce apoptosis in normal liver cells. This is consistent with the results of cell proliferation assays using the CCK-8 assay.

[0112] Western blotting was used to determine the protein expression of BAX and BCL-2 in HepG2 cells after 24 hours of treatment with different concentrations of JUB. The results showed that BAX protein expression was positively correlated with the concentration of JUB treatment (see...). Figure 8 A), and a significant upward trend was observed at 80 μM (see A). Figure 8 B). BCL-2 protein expression was negatively correlated with JUB intervention concentration (see B). Figure 8 C), and showed a significant decrease at 80 μM and 120 μM (see C). Figure 8 D).

[0113] 7.3 JUB can inhibit the migration of liver cancer cells.

[0114] The effect of JUB on the migration of HepG2 liver cancer cells was detected using a cell scratch healing assay. Results are as follows: Figure 9 As shown, after intervention with 80 μM JUB, the cell migration rate of HepG2 cells was significantly reduced (p<0.01), and after intervention with 120 μM JUB, the cell migration rate was significantly inhibited (p<0.01), indicating that high concentrations of JUB intervention can significantly inhibit the migration of liver cancer cells.

[0115] 7.4 JUB can regulate the PI3K / AKT signaling pathway in liver cancer cells.

[0116] Western blotting was used to determine the protein expression of PI3K, p-PI3K, AKT, and p-AKT in HepG2 cells after 24 hours of treatment with different concentrations of JUB. The results showed that PI3K protein expression was positively correlated with the concentration of JUB treatment (see...). Figure 10 A), and a significant upward trend was observed at 80 μM and 120 μM (see A). Figure 10 B). PI3K expression levels also showed a significant upward trend at a 120 μM intervention concentration compared to a 40 μM intervention concentration. p-PI3K protein expression levels were negatively correlated with the JUB intervention concentration (see [link]). Figure 10 C), and a significant decreasing trend was observed at both 80 μM and 120 μM interventions (see C). Figure 10 D). It can be seen that the expression levels of both PI3K and p-PI3K showed significant differences at an intervention concentration of 120 μM. Akt protein expression showed an increasing trend after JUB intervention (see...). Figure 11 A), but there was no significant difference (see Figure 11 B). The expression level of p-Akt protein was negatively correlated with the concentration of JUB intervention (see B). Figure 11 C), and a significant decreasing trend was observed at 120 μM intervention (see Figure 11(D) Notably, the expression level of p-Akt protein at the 120 μM intervention concentration was significantly reduced compared to 40 μM and 80 μM. These results indicate that JUB intervention in HepG2 cells inhibited the PI3K / Akt pathway, thereby regulating BAX and BCL-2 protein expression and ultimately promoting apoptosis.

[0117] 8. Animal experiments to verify

[0118] 8.1 Construction of a SD rat model of liver cancer

[0119] A rat model of liver cancer was established through DEN intervention to evaluate the in vivo antitumor potential of JUB. Figure 12 As shown in Figure A, in the first week, the rat liver was reddish-brown with a smooth, nodule-free surface. In the eighth week, dark black spots appeared on the liver surface, with some areas turning white. In the twelfth week, the liver was enlarged overall, with multiple nodules of varying sizes on the surface, uneven in color, some areas being dark brown with localized yellowing. The tumor nodules were 2-3 mm in diameter, with indistinct borders, and distributed throughout the liver. Blood vessels on the liver surface were significantly dilated, and hemorrhages were visible in some areas. Compared to a normal liver, the model liver was harder, with a rougher surface and obvious pathological changes. In the sixteenth week, the liver was even more enlarged overall, with obvious nodules on the surface. The tumor nodules were 3-5 mm in diameter, with indistinct borders, and distributed throughout the liver. Blood vessel dilation on the liver surface was more pronounced than in the twelfth week, and hemorrhages were visible in some areas. Compared to the liver at the twelfth week, the pathological changes were more pronounced. Figure 12As shown in Figure B, at week four, the liver capsule was composed of dense connective tissue rich in elastic fibers of uniform thickness. The liver lobules were clearly demarcated and regularly arranged. The central vein was located in the center of each lobule, surrounded by hepatocytes and sinusoids arranged in a roughly radial pattern. The hepatocytes were round and plump. The liver plates were regularly and neatly arranged, and there was no obvious dilation or compression of the sinusoids. There were no obvious abnormalities in the portal canal areas between adjacent liver lobules. No obvious inflammatory changes were observed. At week eight, a large number of hepatocytes showed mild fatty degeneration, and small, round vacuoles were visible in the cytoplasm. Mild hemorrhage was commonly seen around the central vein, and a small amount of connective tissue hyperplasia was observed in some areas. At week 12, extensive connective tissue proliferation around portal areas was observed in the liver tissue, forming numerous bridging junctions and pseudolobules, accompanied by scattered lymphocytic infiltration. Numerous bile ducts also showed proliferation and dilation. Many hepatocytes exhibited ballooning degeneration, swelling, centrally located nuclei, and vacuolated cytoplasm. Eosinophilic bodies were rarely seen in the cytoplasm of hepatocytes. Mild fatty degeneration of hepatocytes was common, with small, round vacuoles visible in the cytoplasm. Hemorrhage was frequently observed. At week 16, large clusters of tumor cells were observed in the liver tissue, accompanied by extensive connective tissue proliferation and scattered lymphocytic infiltration. A significant number of hepatocytes showed hydropic degeneration around the tumor, swelling, and loose, pale cytoplasm. A small number of hepatocytes showed fatty degeneration, with round vacuoles of varying sizes visible in the cytoplasm. Ballooning degeneration of hepatocytes was common, with swelling, centrally located nuclei, and vacuolated cytoplasm. Connective tissue proliferation was observed around multiple portal areas, forming bridging junctions and localized pseudolobule formation, accompanied by punctate lymphocytic infiltration. In summary, the DEN-induced liver cancer model was initially established at 12 weeks, and by 16 weeks, liver cancer was clearly visible from the appearance of the liver and HE-stained sections.

[0120] 8.2 Comparison of liver appearance in different groups of rats

[0121] By comparing the appearance of the livers of rats in different groups, the anti-tumor potential of JUB in vivo can be preliminarily observed. Figure 13 Liver A, from the control group, is a uniform reddish-brown color with a smooth, soft surface and thin, sharp edges. It is divided into four lobes, clearly demarcated, and of moderate size, accounting for approximately 4%-5% of body weight. The surface vascular network is evenly distributed, and the bile ducts show no significant dilation. The surface is smooth, uniformly reddish-brown, and no nodules, necrosis, or hemorrhage areas are observed. Liver A, from the DEN group, has an uneven surface with multiple grayish-white nodules of varying sizes, ranging from 2-6 mm in diameter, and a firmer texture. The liver color is uneven, with some areas being dark brown, accompanied by localized hemorrhage and necrosis. The liver edges are blunted, surface blood vessels are significantly dilated, and bile ducts are dilated (see...). Figure 13 B). The number and volume of nodules in the DEN+1mg / g JUB group were reduced compared to the DEN group (see...). Figure 13 C). Compared with the DEN+1mg / g JUB group, the DEN+7mg / g JUB group had fewer nodules, and the color was more reddish (see...). Figure 13 D). In the DEN+14mg / g JUB group, the livers of rats were mildly enlarged, with a generally smooth surface and a few small nodules less than 1 mm in diameter. The livers were slightly uneven in color, with some areas appearing slightly yellow. The liver edges were generally sharp, with slight dilation of surface blood vessels and no obvious dilation of bile ducts (see [link]). Figure 13 E). The results showed that JUB could antagonize DEN-induced hepatocellular carcinoma formation to a certain extent.

[0122] 8.3 Results of HE staining of rat liver

[0123] The antitumor potential of JUB in vivo was assessed by HE staining. Figure 14 As shown in Figure A, the liver capsule of the control group rats consisted of dense connective tissue rich in elastic fibers of uniform thickness. The liver lobules were clearly demarcated and regularly arranged. A central vein was located in the center of each lobule, surrounded by hepatocytes and sinusoids arranged in a roughly radial pattern. The hepatocytes were round and plump. The liver plates were regularly and neatly arranged, and the sinusoids showed no obvious dilation or compression. There were no obvious abnormalities in the portal areas between adjacent liver lobules. No obvious inflammatory changes were observed. Locally, numerous hepatocytes were enlarged, with increased basophilicity in the cytoplasm. A small number of eosinophilic bodies were visible in the cytoplasm, along with punctate hepatocyte necrosis and nuclear fragmentation. A large number of portal ducts showed bile duct hyperplasia with dilation, and connective tissue hyperplasia forming multiple bridging points. Local pseudolobules were formed, accompanied by scattered lymphocyte infiltration (see Figure A). Figure 14 B). In the DEN+1mg / gJUB group, hepatocyte hydropic degeneration, cell swelling, and loose, pale cytoplasm were commonly observed in the liver tissue. Mild fatty degeneration of hepatocytes was also frequently seen, with small, round vacuoles visible in the cytoplasm. Locally, ballooning degeneration of hepatocytes was observed, with cell swelling, centrally located nuclei, and cytoplasmic vacuolation. Connective tissue hyperplasia was observed in numerous portal areas, accompanied by bile duct hyperplasia. Locally, numerous tumor cells were observed, accompanied by hemorrhage, as well as substantial connective tissue hyperplasia and significant lymphocyte infiltration (see...). Figure 14 C). In the liver tissue of rats in the DEN+7mg / g JUB group, a large number of hepatocytes showed hydropic degeneration, cell swelling, and loose, pale cytoplasm. Mild fatty degeneration of hepatocytes was common, with small, round vacuoles visible in the cytoplasm. Ballooning degeneration of hepatocytes was less common, with cell swelling, centrally located nuclei, and cytoplasmic vacuolation. Multiple bile ducts were observed. Connective tissue hyperplasia was rare, and punctate lymphocyte infiltration was observed (see...). Figure 14 D). In the liver tissue of rats in the DEN+14mg / g JUB group, a large number of hepatocytes showed hydropic degeneration, cell swelling, loose and pale cytoplasm, and mild fatty degeneration of a large area of ​​hepatocytes. Small round vacuoles were visible in the cytoplasm. Ballooning degeneration of hepatocytes was also observed, with cell swelling, centrally located nuclei, and cytoplasmic vacuolation. Mild bile duct hyperplasia was observed in multiple portal areas, with local dilation. Mild connective tissue hyperplasia and punctate lymphocyte infiltration were also observed (see D). Figure 14E). The results showed that JUB could antagonize DEN-induced hepatocellular carcinoma formation to a certain extent.

[0124] 8.4 Changes in rat body weight

[0125] During the establishment of the rat model of liver cancer and the JUB intervention, the rats were weighed every four days to observe changes in body weight and whether there were significant differences in body weight among different groups. As shown in the figure below, the body weight of each group of rats was positively correlated with the feeding time, with the control group showing faster weight gain. On day 28, the DEN group and the DEN+JUB (1 mg / g) group showed significant differences compared to the control group, meaning that the body weight of the control group rats was significantly higher than that of the DEN group and the 1 mg / g JUB intervention group from day 28. On day 68, the body weight of the control group rats also showed a significant difference compared to the DEN+JUB (7 mg / g) group, meaning that from day 68 onwards, the body weight of the other three groups of rats, except for the DEN+JUB (14 mg / g) group, was significantly lower than that of the control group. It is noteworthy that the body weight of the DEN+JUB (14 mg / g) group rats was never significantly lower than that of the control group rats (see Figure 1). Figure 15 This result indicates that JUB can significantly antagonize the decrease in body weight in rats during liver cancer formation.

[0126] 8.4 Feeding status of rats

[0127] During the establishment of the rat model of liver cancer and JUB intervention, the food intake of rats was measured weekly to observe whether there were significant differences in food intake among different groups during the modeling intervention. As shown in the figure below, the average weekly food intake of rats in the DEN group and the different concentrations of JUB intervention groups was significantly lower than that of the control group, while the DEN+JUB (7 mg / g) and DEN+JUB (14 mg / g) groups were significantly higher than that of the DEN group. It is worth mentioning that the average weekly food intake of rats in the DEN+JUB (14 mg / g) group was also significantly higher than that of the DEN+JUB (1 mg / g) group (see figure below). Figure 16 The results showed that JUB significantly increased food intake in rats during the process of liver cancer formation.

[0128] 8.5 Changes in the visceral index of rats

[0129] After 16 weeks of intervention, rats were sacrificed, and their livers were removed, weighed, and compared with their body weight to obtain the hepatic visceral index. As shown in the figure below, compared with the control group, the visceral index of each group showed an increasing trend, except for the DEN group, which showed a significant increase compared with the control group (see figure). Figure 17 The results indirectly indicate that JUB can significantly inhibit the formation of liver cancer or hepatic proliferative lesions in SD rats.

[0130] 8.5 Effects of JUB on the pathway protein PI3K / Akt in rat liver tissue

[0131] Depend on Figure 18 It was found that there was no significant difference in the expression level of PI3K protein among the groups. However, the expression level of Akt protein in the DEN+JUB (1 mg / g) and DEN+JUB (7 mg / g) groups was significantly higher than that in the control group (see...). Figure 19 In the four groups other than the control group, the expression level of p-PI3K protein was negatively correlated with the intervention concentration of JUB, and a significant decrease was observed at intervention levels of 1 mg / g, 7 mg / g, and 14 mg / g. The DEN+JUB (7 mg / g) and DEN+JUB (14 mg / g) groups were significantly lower than the DEN+JUB (1 mg / g) group, and there was no significant difference compared to the control group (see...). Figure 20 The expression levels of p-Akt protein in each group are as follows: Figure 21 As shown, the DEN and DEN+JUB (1 mg / g) groups were significantly higher than the control group. In the other four groups besides the control group, the expression level of p-AKT protein was negatively correlated with the intervention concentration of JUB, and a significant decrease was observed at intervention levels of 7 mg / g and 14 mg / g. The DEN+JUB (7 mg / g) and DEN+JUB (14 mg / g) groups were also significantly lower than the DEN+JUB (1 mg / g) group, but there was no significant difference compared to the control group. These results indicate that after JUB intervention in hepatocellular carcinoma SD rats, the activation proteins p-PI3K and p-AKT of PI3K and Akt in the liver tissue of hepatocellular carcinoma rats were inhibited, consistent with the trend observed in the cell experiments.

[0132] 8.6 Effects of JUB on apoptosis-related proteins BAX and BCL-2 in rat liver tissue

[0133] from Figure 22 The results showed that the expression level of BAX protein was positively correlated with the intervention concentration of JUB, and the expression level of BAX protein in the DEN+JUB (7 mg / g) and DEN+JUB (14 mg / g) groups was significantly higher than that in the control group and the DEN group (see [link to data]). Figure 22 F). By Figure 23 It was found that the BCL-2 protein expression level in the DEN group was significantly higher than that in the control group. In the other four groups besides the control group, the BCL-2 protein expression level was negatively correlated with the intervention concentration of JUB, and the BCL-2 protein expression level in the DEN+JUB (1 mg / g), DEN+JUB (7 mg / g), and DEN+JUB (14 mg / g) groups was significantly lower than that in the control group and the DEN group (see...). Figure 23 F). The results showed that after JUB intervention in rats with liver cancer, the expression level of BAX protein increased while the expression level of BCL-2 protein decreased, promoting apoptosis of liver cancer cells, which was consistent with the trend in the cell experiment section.

[0134] In summary, JUB intervention in rats with hepatocellular carcinoma inhibited the PI3K / Akt pathway, thereby regulating the expression of BAX and BCL-2 proteins and ultimately promoting apoptosis of rat hepatocellular carcinoma cells.

Claims

1. The application of jujube seed in the preparation of anti-liver cancer drugs, characterized in that, The active ingredient in the jujube seed is jujube seed saponin B.

2. The method for analyzing the mechanism of action of the application as described in claim 1, characterized in that, Includes the following steps: S1. Screening and target prediction of active ingredients in jujube seed: Information on bioactive components in jujube seed was obtained by screening the TCMSP database, and then the chemical formulas and PubChem_CIDs of the above bioactive components in jujube seed were obtained by using the PubChem database. Using the SwissTargetPrediction database, potential targets of action were identified based on the chemical formulas of the canonical smiles of bioactive components, with humans selected as the species. Simultaneously, the targets were screened using the BATMAN-TCM database based on Pubchem_CID. The bioactive components and targets obtained from the two databases were combined to obtain the targets of action of the bioactive components of jujube seed. S2. Liver cancer target screening: Using the GeneCards database, the keyword "hepatocellular carcinoma" was entered to retrieve liver cancer targets related to liver cancer. The intersection of liver cancer targets and the targets of active ingredients in jujube seed was obtained to obtain the anti-liver cancer targets of active ingredients in jujube seed. S3. Pathway enrichment analysis: The active ingredients of Ziziphus jujuba seed were input into the DAVID database for GO and KEGG pathway enrichment analysis, and the PI3K-AKT signaling pathway was significantly enriched. S4.PPI Network Analysis: The active ingredients of Ziziphus jujuba seed were input into the STRING database to obtain the interaction information between the active ingredients of Ziziphus jujuba seed and the anti-liver cancer target; the Cytohubba plugin in Cytoscape software was used to perform molecular analysis of key targets to obtain the key target molecule AKT1. S5. Molecular docking: Molecular docking was performed between the bioactive components information of jujube seed and key target molecules, revealing that the interaction between jujube seed saponin B and the key target molecule AKT1 is closely related to liver cancer. S6. Biological verification: It was confirmed that jujube seed saponin B has an anti-liver cancer mechanism.

3. The mechanism of action analysis method according to claim 2, characterized in that: In step S1, 33 active ingredients of jujube seed were screened using TCMSP; the canonical smiles of the bioactive substances were searched through the Pubchem database, and then the targets of the active substances were searched through the SwissTargetPrediction database. After removing duplicate targets, 28 active substances and 751 targets were obtained; the Pubchem CID of the bioactive substances was searched through the Pubchem database, and then the targets of the active substances were searched through the BATMAN-TCM database, resulting in 27 active ingredients and 580 targets; finally, the intersection of the targets obtained from the two databases yielded 26 bioactive ingredients of jujube seed and 144 corresponding targets.

4. The mechanism of action analysis method according to claim 3, characterized in that: In step S2, a set of liver cancer-related genes was screened through the GeneCards database. Based on the keyword "hepatocellular carcinoma", a total of 8396 genes were obtained. The intersection of 144 target molecules with the 8396 liver cancer target molecules yielded 105 identical genes, which are the anti-liver cancer target molecules of jujube seed active ingredients.

5. The mechanism of action analysis method according to claim 2, characterized in that: In step S3, the 105 active ingredients of jujube seed obtained from the above analysis were subjected to GO and KEGG enrichment analysis using the DAVID database. The corrected p < 0.05 was used as the screening condition in the results.

6. The mechanism of action analysis method according to claim 2, characterized in that: In step S6, the biological verification includes cell experiment verification and animal experiment verification.

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