Method for analyzing action mechanism of external medicine for treating liver cancer ascites

Abstract

The invention discloses an action mechanism analysis method of an external medicine for treating liver cancer ascites, and relates to the technical field of medicines. Comprising the following steps: screening active ingredients and related targets of a medicine through network pharmacology; predicting liver cancer ascites related disease targets; constructing an intersection target point network of active ingredient target points and disease target points, and performing protein interaction network analysis; carrying out GO function and KEGG pathway enrichment analysis on the intersection target spot; establishing a medicine, component, disease, target spot and pathway network; the binding activity of the core target and the active component is verified through molecular docking; establishing a liver cancer ascites model through animal experiments, and performing efficacy verification; network pharmacology and animal experiment data are integrated, and a drug action mechanism is analyzed. According to the invention, active ingredients, target spots and pathways of the medicine are rapidly screened through network pharmacology, so that the earlier-stage research period is greatly shortened; animal experiment design is standardized, samples can be processed in batches, and the method is suitable for high-throughput drug screening.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for analyzing the mechanism of action of a topical drug in treating ascites caused by liver cancer. Background Technology

[0002] Liver cancer is one of the most common and most dangerous malignant tumors worldwide. Among the five major complications of liver cancer, malignant ascites has the highest incidence. The presence of ascites indicates rapid disease progression, difficult treatment, poor quality of life, short survival, and a dangerous prognosis. Therefore, the treatment of ascites is an important part of alleviating the condition of patients with advanced liver cancer.

[0003] Currently, Western medicine treatment for ascites is mainly based on the principle of promoting ascites drainage. Common methods include the use of potassium chloride diuretics, albumin supplementation, abdominal drainage, and peritoneal dialysis. Because ascites caused by liver cancer is malignant and prone to recurrence, treatment can only relieve ascites symptoms, with generally limited effectiveness, a long treatment course, and a risk of infection.

[0004] Traditional Chinese medicine (TCM) believes that the basic pathological mechanism of ascites in liver cancer is "insufficient function of the liver, spleen, and kidneys, leading to the stagnation of qi, blood, and fluids in the abdomen." Oral decoctions combined with methods to enhance the body's resistance and eliminate pathogenic factors are remarkably effective in treating ascites. However, some patients may refuse to take the decoctions due to abdominal distension, nausea, or concerns that excessive fluid intake will increase ascites, which can affect the treatment outcome. Reports indicate that external application of TCM can improve ascites symptoms and has the advantages of simple preparation, rapid absorption, and good patient compliance.

[0005] This study explores the therapeutic effects of external application of traditional Chinese medicine on ascites caused by liver cancer. Simultaneously, network pharmacology is used for target analysis to investigate its molecular mechanism of action, providing a theoretical basis for better clinical application of external application of traditional Chinese medicine. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for analyzing the mechanism of action of topical drugs in treating ascites in liver cancer.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for analyzing the mechanism of action of a topical drug in treating ascites caused by liver cancer, wherein the preparation method of the topical drug includes the following steps: S1: Prepare the loofah fibers with skin on, which include: loofah sponge, croton seeds, and white rice; S2: Place the croton seeds into the loofah sponge, stir-fry over low heat until the loofah sponge turns a dark yellow color, then remove it and remove the croton seeds; S3: Stir-fry the rice and loofah over low heat until the rice turns a dark yellow color and the loofah turns completely black. S4: Grind the above ingredients into powder and mix with glycerin at a mass ratio of 1:1.5; S5: Apply to gauze to obtain a topical medication dressing.

[0008] Preferred: Its active ingredients include at least one of phloretin, decanoic acid, lauric acid, caprylic acid, and croton toxin.

[0009] A method for analyzing the mechanism of action of a topical drug in treating ascites in liver cancer includes the following steps: S1: Screening drug active ingredients and their related targets through network pharmacology; S2: A disease target for predicting ascites-related liver cancer; S3: Construct an intersection target network of active ingredient targets and disease targets, and perform protein-protein interaction network analysis; S4: Perform GO function and KEGG pathway enrichment analysis on the intersection targets; S5: Establish a network of drugs, ingredients, diseases, targets, and pathways; S6: Verify the binding activity between the core target and the active ingredient through molecular docking; S7: Establish a liver cancer ascites model through animal experiments to verify the efficacy of the drug; S8: Integrate network pharmacology and animal experimental data to analyze drug mechanisms of action.

[0010] Preferably, in step S1, the active ingredient screening uses the TCMSP database, with the following parameters set: molecular weight ≤ 500, number of hydrogen bond donors ≤ 5, number of hydrogen bond acceptors ≤ 10, lipid-water partition coefficient between -2 and 5, and number of rotatable bonds ≤ 10.

[0011] Preferably, in step S1, the target prediction uses the SwissTargetPrediction database with a probability threshold ≥0.1, and gene symbol conversion is performed using the UniProt database.

[0012] Preferably, in step S3, the intersection target network is constructed using the Venny tool, the PPI network is constructed using the STRING database with a confidence level > 0.7, and topology analysis is performed using Cytoscape software. The screening criteria include degree centrality, proximity centrality, and betweenness centrality all being greater than the median.

[0013] Preferably, in S7, the animal experiment uses the Kunming mouse ascites model induced by H22 liver cancer cells, and the groups include a blank group, a model group, a positive drug group, and a drug treatment group; the drug treatment group includes low-dose and high-dose external application treatment.

[0014] Preferably, in S7, the method for verifying drug efficacy includes detecting mouse abdominal circumference, ascites volume, serum ALT, AST, and ALB levels, liver tissue pathological HE staining, and liver tissue IL-6, AKT1, ESR1, and JUN protein expression.

[0015] Preferably, the mechanism of action is a synergistic effect of multiple targets and pathways, wherein the targets include IL-6, AKT1, ESR1 and JUN, and the pathways include cancer pathways, hepatitis B pathways and viral oncogenic pathways.

[0016] The beneficial effects of this invention are as follows: 1. This invention rapidly screens drug active ingredients, targets and pathways through network pharmacology, significantly shortening the preliminary research cycle; the animal experiment design is standardized, and samples can be processed in batches, making it suitable for high-throughput drug screening.

[0017] 2. This invention utilizes PPI networks, GO / KEGG enrichment analysis, and other methods to comprehensively reveal the synergistic mechanism of drugs through multiple pathways such as key targets like IL-6, AKT1, ESR1, and JUN, as well as cancer pathways and hepatitis B pathways, avoiding the shortcomings of traditional methods that only focus on a single target.

[0018] 3. The present invention uses molecular docking to verify the binding activity of the core target. Animal experiments directly verify the predicted results through serum indicators, histopathology and protein expression detection (such as ALT, AST, ALB, IL-6, etc.), forming a two-way verification between calculation and experiment, and improving the credibility of the conclusions.

[0019] 4. This invention can effectively reduce the amount of ascites and abdominal circumference in mice with liver cancer ascites model, and improve the overall condition of the animals. Animal experiments showed that compared with the model group, the abdominal circumference and amount of ascites in the drug treatment group (especially the low-dose group) were significantly reduced (P<0.01), and the low-dose group was more effective than the positive control group (cyclophosphamide). The mice in the model group showed irritability, reduced food intake and dull fur, while the mice in the treatment group showed improved mental state, glossy fur and increased food intake, indicating that the drug can alleviate the systemic adverse effects of the disease.

[0020] 5. The drug of this invention can significantly improve liver function indicators and reduce liver tissue damage through external application. Serum tests showed that the AST and ALT levels were increased and the ALB level was decreased in the model group, while the AST and ALT activities were decreased and the ALB level was increased in the treatment group (P<0.05 or P<0.01), indicating that albumin synthesis capacity was restored and hepatocyte damage was reduced. Liver morphology observation showed that the liver size, color and texture of the treatment group were improved compared with those of the model group, and liver lobe adhesions were reduced.

[0021] 6. Network pharmacology and molecular docking validation showed that the drug acts on core targets such as IL-6, AKT1, ESR1, and JUN through multiple components (such as croton toxin and phloretin), achieving multi-pathway synergistic regulation. In animal experiments, the expression of pro-inflammatory and pro-cancer proteins IL-6, AKT1, and JUN was downregulated in the treatment group, while the expression of the protective protein ESR1 was upregulated (P<0.01), which is consistent with the network prediction results. Molecular docking showed that the binding energy between the active ingredient and the target was ≤0 kJ / mol, confirming good binding activity, such as the interaction between croton toxin and targets such as EP300 and PTGS2. Attached Figure Description

[0022] Figure 1 This is a Venny analysis diagram of the drug-disease intersection target of the present invention; Figure 2 This is a panoramic view of the protein interaction network of the present invention; Figure 3 This is a topological analysis diagram of the core target points of this invention; Figure 4 This is a bar chart showing the GO functional enrichment analysis of the present invention. Figure 5 This is a bubble diagram of KEGG pathway enrichment analysis according to the present invention. Figure 6 This is a drug-component-disease-target-pathway network diagram of the present invention; Figure 7 These are comparative images of the animal models used in this invention. Figure 8 This is a comparative image of the gross morphology of the liver in this invention; Figure 9 This is a liver tissue pathology HE staining image of the present invention; Figure 10 This is a statistical chart of serum liver function indicators according to the present invention; Figure 11 This is a Western blotting diagram showing the expression of the key protein in this invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: A method for analyzing the mechanism of action of a topical drug in treating ascites caused by liver cancer, as detailed below: I. Materials Drugs and reagents: Mouse hepatocellular carcinoma cells (H22) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China, SCSP-515, TCM13). H22 cells were incubated in RPMI 1640 medium (DMEM, Gibio, USA) containing 10% FBS and 1% penicillin-streptomycin. All cells were incubated at 37°C under a humidified environment of 5% CO2. ALT / GPT assay kit, AST / GOT assay kit, and albumin assay kit (Nanjing Jiancheng Biotechnology Institute, batch numbers C009-2-1, C010-2-1, A028-2-1, respectively); PMSF, RIPA lysis buffer, Bradford protein assay kit, phosphatase inhibitor mixture A (50×), protease inhibitor mixture (100×), and SDS-PAGE protein loading buffer (5×) (Beyotime, batch number ST506, respectively). P0013B, P0006C, P1081, P1005, P0015L); protein loading buffer, protein-free rapid blocking buffer (5×) (Abbkine, batch numbers LT103 and PS108 respectively); Superkine™ high-sensitivity ECL chemiluminescence solution (Finker grade) (Abbkine, batch number BMU102-CN); β-actin (Servicebio, batch number GB15003), rabbit anti-AKT1, rabbit anti-il-6, rabbit anti-ESR1, rabbit anti-C-JUN, rabbit anti-HSP90 alpha, HRP-labeled goat anti-rabbit secondary antibody (Affinit Bioscience, catalog numbers AF0836, DF6087, AF6058, AF6090, AF5368, S0001).

[0025] Instruments: Synergy fully automated multifunctional microplate reader (Hong Kong Gene Technology Co., Ltd.); optical microscope (Olympus Corporation, Japan); BG-verMINI mini vertical electrophoresis apparatus (Beijing Baijing Biotechnology Co., Ltd.) II. Methods Screening and target prediction of components in loofah fibers with skin: Active components and related targets of the skin-on loofah fiber were screened in the TCMSP database (https: / / www.tcmsp-e.com / ) based on the following criteria: (MW) ≤ 500, number of hydrogen bond donors (Hdon) ≤ 5, number of hydrogen bond acceptors (Hacc) ≤ 10, lipid-water partition coefficient (AlgP) between -2 and 5, and number of rotatable bonds (RBN) ≤ 10. Target prediction was performed using the SwissTargetPrediction database (http: / / swisstargetprediction.ch / ) (probability ≥ 0.1), and UniProt (https: / / www.uniprot.org / ) converted the target protein names into gene symbols.

[0026] Prediction of target in ascites in liver cancer: The keyword "liver cancer ascites" was entered into the GeneCards (https: / / www.genecards.org), OMIM (https: / / omim.org / ), and TTD (https: / / db.idrblab.net / ttd / ) databases to search for relevant gene targets, and duplicate entries were integrated and removed.

[0027] Construction of Venny diagrams and protein-protein interaction networks: Intersection target points were obtained using venny2.1.0 (https: / / bioinfogp.cnb.csic.es / ) and imported into the STRING database (http: / / string-db.org). With "highest confidence" > 0.7, disconnected nodes in the network were hidden. After importing into Cytoscape 3.9.1 software, multi-parameter topology analysis was performed using the CytoNCA plugin (Degree centrality (DC), Closeness centrality (CC), and Betweenness centrality (BC) were all > median) to screen core target points.

[0028] GO function and KEGG pathway enrichment analysis: Intersecting targets were imported into the David database (http: / / david.ncifcrf.gov / ) for GO function and KEGG pathway enrichment analysis, and the data were uploaded to Bioinformatics (http: / / www.bioinformatics.com.cn / ) for visualization.

[0029] Construction of the drug-component-disease-target-pathway network: The screened active ingredients, disease targets, overlapping targets, and KEGG pathway data are integrated into Cytoscape software, and a drug-ingredient-disease-target-pathway network is built using the CytoNCA plugin. The node size varies according to the degree value.

[0030] Molecular docking verification: AutoDock Vina was used to perform molecular docking between the core targets and major active ingredients in the PPI network. Finally, the results were visualized using Discovery Studio 2019 Client software.

[0031] Animal experiments: After 3 days of acclimatization, a mouse model of liver cancer with ascites was established according to the literature. Mice were randomly divided into the following 5 groups: blank group, model group, positive drug group (cyclophosphamide), low-dose group with attached loofah sponge, and high-dose group with attached loofah sponge, with 10 mice in each group. H22 cell lines were revived in a 37℃ water bath at 1000 rpm. -1 After centrifugation for 5 minutes, the cell pellet was resuspended in physiological saline to a cell count of approximately 1.0 × 10⁶ cells / min. 7 A mouse model of H22 liver cancer ascites was established by intraperitoneal injection of 0.2 mL of the vaccine per mL. The mice were observed after inoculation and were housed in an SPF-grade animal laboratory.

[0032] Modeling was successful one week later, and drug administration began on the second day for 7 consecutive days. The control group and model group received topical patches containing only glycerin, while the positive control group (cyclophosphamide) received a single dose of 100 mg / kg. -1 Cyclophosphamide injection was administered to the low-dose group and the high-dose group of the loofah sponge with skin, respectively, with 1cm×1cm and 2cm×2cm external application patches, and the administration was continued for one week.

[0033] Monitor the general condition of mice in each group, including appearance, activity and mental state. Before taking samples at the end of the experiment, measure the abdominal circumference (cm) of the mice. After enucleating the eyeballs to collect blood, extract ascites and measure the ascites volume (ml).

[0034] Blood was collected from the eyeballs, allowed to stand for 30 minutes, and then centrifuged at 3500 rpm for 10 minutes at 4°C. The supernatant was collected. Serum AST, ALT, and ALB levels in each group of mice were measured according to the kit instructions.

[0035] The liver was isolated, and the morphology of the livers in each group was compared and photographed. A portion of the mouse liver was carefully removed intact and fixed in 4% paraformaldehyde fixative. It was then placed at room temperature for HE staining (dewaxing, staining, dehydration, clearing, and mounting) to demonstrate the basic morphology of the liver tissue.

[0036] The remaining liver tissue was rapidly frozen in liquid nitrogen and stored at -80°C for Western blot analysis to determine protein expression levels. 60 mg of each sample was weighed, and 600 μL of lysis buffer was added to each tube. After low-temperature grinding, the mixture was incubated at 4°C and 12000 rpm. -1 Centrifuge for 10 min, collect the supernatant, determine the protein concentration of each sample using a Bradford reagent kit, add loading buffer, boil in water for 10 min, aliquot and store at -20℃. For SDS-PAGE gels, load 30 μg per well, upper gel voltage 60 V, lower gel voltage 120 V, electrophoresis until bromophenol blue reaches the bottom of the gel, transfer membrane using a constant current of 300 mA for 30 min, block with 5% BSA at room temperature for 1 h, add primary antibody and incubate overnight at 4℃; the next day, wash 5 times with TBST for 5 min each time, add corresponding secondary antibody and incubate at room temperature for 1 h, wash again with TBST, add chemiluminescence solution, and image in a chemiluminescence system. Results are expressed as grayscale values.

[0037] Statistical analysis: The experimental data were analyzed using GraphPad Prism 9.5.1 statistical software. One-way ANOVA was used for comparisons among multiple groups to test whether they conformed to normal distribution and homogeneity of variance. Non-parametric tests were performed for variance inequality. Data are expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant.

[0038] III. Results Screening of active ingredients: Thirteen components of the fiber from the skin-on loofah were screened from the TCMSP database. After prediction and duplicate removal from the SwissTargetPrediction database, a total of 251 potential targets were obtained. See the table below for details: Number MolID name MW AlogP Hdon Hacc RBN 1 MOL001624 Phloretol 274.29 2.66 4 5 4 2 MOL001626 Phrymarolin-II 458.45 2.3 0 10 6 3 MOL001627 Tropigline 223.35 2.2 0 3 3 4 MOL001628 Crotin (chalcone) 340.4 3.71 3 5 4 5 MOL001629 Crotonic acid 86.1 0.87 1 2 1 6 MOL001639 Tiglic acid 100.13 1.32 1 2 1 7 MOL001640 Capric Acid 172.3 3.63 1 2 8 8 MOL001643 Croweacin 192.23 2.59 0 3 3 9 MOL001644 Dodecanal 184.36 4.59 0 1 10 10 MOL000261 Myristicin 192.23 2.59 0 3 3 11 MOL000303 Octanoic Acid 144.24 2.72 1 2 6 12 MOL000305 Lauric acid 200.36 4.54 1 2 10 13 MOL000475 anethole 148.22 2.77 0 1 2 Prediction of target in ascites in liver cancer: A total of 1655 targets were obtained by searching the GeneCards, OMIM, and TTD databases for targets related to ascites in liver cancer and removing duplicates.

[0039] Venny graphs and PPI networks: The Venny plot shows 73 intersecting target points ( Figure 1 Import it into the String database and create a PPI network diagram. Figure 2 ), followed by topology analysis ( Figure 3 The result includes 66 nodes and 326 edges.

[0040] GO and KEGG enrichment analysis: GO enrichment yielded 452 entries. These included 284 biological processes (BP), 55 cellular components (CC), and 113 molecular functions (MF), which were imported into the David website (P < 0.05). Analysis showed that molecular functions mainly involved transcriptional coactivator binding, nuclear receptor activity, and protein binding. Cellular components primarily covered the nucleoplasm, cyclin-dependent protein kinase holoenzyme complex, and cytoplasm. Biological processes mainly involved cellular responses to lipopolysaccharide, positive regulation of transcription by RNA polymerase II, positive regulation of the MAPK cascade, and positive regulation of gene expression. Figure 4 KEGG enrichment results identified 94 signaling pathways, including key pathways such as cancer pathways, hepatitis B pathways, viral oncogenic pathways, and chemical oncogenic-receptor activation pathways. Figure 5 ).

[0041] Drug-Ingredient-Disease-Target-Pathway Screening Results: Establish a drug-component-disease-target-pathway network diagram ( Figure 6 This visually demonstrates the mechanism by which the skin-on loofah fiber intervenes in ascites caused by liver cancer through multiple targets and pathways. The parallelograms, hexagons, and squares represent the active ingredient, signaling pathway, and target, respectively.

[0042] Molecular docking results: Molecular docking was performed on the top-ranked active ingredients and molecular targets based on degree centrality (DC). The results showed that the binding energies of these ligands were all ≤0 kJ / mol, indicating that they could bind spontaneously, as detailed in the table below: Molecular docking between molecular targets and active ingredients:

[0043] In vivo experimental verification: General condition of mice in each group, changes in abdominal circumference and ascites volume: General animal condition: Mice in the control group showed normal mental state, with no abnormalities in fur color or appetite. Mice in the model group exhibited restlessness, significantly reduced food intake, dull fur, and decreased activity. After drug treatment, the basic condition of mice in all treatment groups improved, showing improved mental state, glossy fur, and increased food intake. Figure 7 .

[0044] Compared with the control group, the model group significantly increased abdominal circumference and ascites volume in mice (P < 0.01). Compared with the model group, the positive control group, the low-dose group and the high-dose group of the sponge with skin all reduced abdominal circumference and ascites volume in mice to varying degrees (P < 0.01), with the low-dose sponge with skin showing a better effect than the positive control group. These results were statistically significant (P < 0.01). See the table below.

[0045] Observation of liver morphology: The livers of mice in the normal group were fuller, with smooth capsules, a dark red and glossy color, and neat, intact edges. The livers of mice in the model group were significantly smaller than those in the normal group, with a more taut capsule, a harder texture, a dark brown color, and a noticeably rough surface. Extensive adhesions were present between the liver and omentum, and between liver lobes (see...). Figure 8 The livers of the mice in the treatment group were between those of the normal group and the model group. The livers were dark red, with a rough texture and blunt edges. There were some adhesions between the liver lobes.

[0046] HE staining results of mouse peritoneal tissue: In the normal group, the liver tissue structure of mice was clear, the hepatocytes were uniform in size and morphology, arranged in cords and radially distributed around the central vein, and no abnormalities were observed in the portal area. In the model group, the hepatocytes of mice were significantly swollen and disordered, with varying degrees of degeneration and necrosis, extensive inflammatory cell infiltration, and enlarged portal area. In the treatment group, the liver damage of mice was reduced to varying degrees compared with the model group, with some hepatocytes showing degeneration, necrosis, and swelling, and some inflammatory cell infiltration was visible in the portal area. Figure 9 ).

[0047] Effects of serum from different groups of mice: Compared with the control group, the serum AST and ALT levels in the model group were significantly increased, while the ALB level was decreased. Compared with the model group, the serum AST and ALT levels in the treatment group were decreased to varying degrees, while the ALB level was increased. Results are shown in [link to results]. Figure 10 .

[0048] Effects of IL-6, JUN, ESR1, and AKT1 protein expression in liver tissue of mice with hepatocellular carcinoma and ascites Compared with the control group, the expression of IL-6, JUN, and AKT1 proteins and the expression of ESR1 protein were increased and decreased in the liver tissue of mice in the model group (P < 0.01). Compared with the model group, the expression of IL-6, JUN, and AKT1 was decreased and the expression of ESR1 was increased in the low-dose group (P < 0.01). The expression of IL-6, JUN, and AKT1 proteins and the expression of ESR1 were decreased and increased in the liver tissue of mice in the positive group and the high-dose group with attached loofah tendon (P < 0.05, P < 0.01). Figure 11 .

[0049] IV. Conclusion: Malignant ascites is a significant complication in the progression of advanced hepatocellular carcinoma, serving as a prognostic biomarker associated with disease progression, treatment resistance, and decreased survival. It is primarily caused by chronic liver dysfunction, leading to reduced albumin synthesis, decreased plasma colloid osmotic pressure, or portal hypertension, resulting in an imbalance of intracellular and extracellular water metabolism. External application of medication to treat ascites is a characteristic therapy in traditional Chinese medicine. This involves applying ointments or pastes to specific acupoints on the abdomen, utilizing the skin for absorption to achieve therapeutic effects. This study, based on network pharmacology, used computer simulations and multiple databases to screen compounds and targets from the skin of a loofah sponge, constructing a "compound-target-disease" network. This provides new ideas and methods, revealing a multi-component, multi-target mechanism of action, and exploring the mechanism of action of externally applied loofah sponge in treating ascites in hepatocellular carcinoma. The aim is to provide innovative concepts for the treatment of ascites in hepatocellular carcinoma and to explore new therapeutic targets.

[0050] Network pharmacology screening identified 13 active ingredients and 73 common targets. Ingredients with high degree values ​​included phlorizin, decanoic acid, lauric acid, caprylic acid, and loofah fiber toxin. High-degree targets included IL-6, AKT1, ESR1, and JUN, suggesting these may be the main active ingredients and key targets of loofah fiber in improving ascites in liver cancer. Molecular docking demonstrated good affinity. KEGG analysis results may involve key pathways such as cancer pathways, hepatitis B pathways, and viral oncogenic pathways, which may be the main pathway by which loofah fiber prevents and treats ascites in liver cancer.

[0051] To validate the network pharmacology analysis results, an animal model of hepatocellular carcinoma (HCC) ascites was established, and serum liver function indicators were measured. Results showed that compared to the treatment group, the model group had significantly increased AST and ALT activities in serum, while ALB levels were significantly decreased. Further pathological examination of the liver revealed that hepatocytes in the model group exhibited significant swelling, disordered arrangement, and varying degrees of degeneration, necrosis, and extensive inflammatory cell infiltration. Furthermore, detection of IL-6, AKT1, JUN, and ESR1 protein expression in liver tissue showed that in the HCC ascites model, the expression of IL-6, AKT1, and JUN was upregulated, while ESR1 expression decreased. After drug treatment, except for a slight increase in ESR1 expression, the expression of the other three proteins decreased.

[0052] In mice treated with high-dose loofah sponge (with skin attached), some developed skin redness and blistering, while the low-dose group showed better therapeutic effects. The skin, as a barrier, has a saturation point for drug absorption. Exceeding this limit, excess drug components not only fail to be effectively absorbed but also accumulate on the skin surface, exacerbating irritation. Low-dose loofah sponge allows for gentle and sustained drug penetration, avoiding burns or excessive inflammatory reactions, and is highly safe. High doses, on the other hand, with excessively strong stimulation, may directly damage tissue and worsen the condition.

[0053] In summary, the possible mechanism by which the skin-on loofah tendon improves ascites in liver cancer is the synergistic effect of multiple components, multiple targets, and multiple pathways, which may be related to IL-6, AKT1, JUN, and ESR1 proteins, providing a theoretical basis for better clinical application of the skin-on loofah tendon external patch.

[0054] Example 2: A topical medication for treating ascites in liver cancer, the preparation method of which includes the following steps: S1: Prepare the following ingredients: 4 ounces of loofah sponge, 3 mace of croton seeds, and 1 liter of white rice; S2: Place the croton seeds into the loofah sponge, stir-fry over low heat until the loofah sponge turns a dark yellow color, then remove it and remove the croton seeds; S3: Stir-fry one liter of rice with loofah sponge over low heat until the rice is old and yellow and the loofah sponge is black and translucent. S4: Grind into powder and mix with glycerin at a mass ratio of 1:1.5; S5: Apply to gauze for dressing.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A topical medication for treating ascites caused by liver cancer, characterized in that, The external medicine preparation method comprises the following steps: S1: prepare the continuous skin luffa tendon: luffa, croton, white rice; S2: put the croton into the luffa by grains, fry the luffa to old yellow color with a small fire, then take out and remove the croton; S3: fry the rice with the luffa again with a small fire until the rice is old yellow and the luffa is black; S4: grind the above ingredients into powder, and mix with glycerin with a mass ratio of 1:1.5; S5: apply on gauze to obtain the external medicine patch.

2. The external medicine for treating liver cancer ascites according to claim 1, characterized in that, The active ingredients include at least one of phloretin, n-decanoic acid, lauric acid, octanoic acid and croton toxin.

3. A method for analyzing the mechanism of action of an external drug in treating liver cancer ascites, characterized in that, The method comprises the following steps: S1: screening active ingredients of the medicine and related targets through network pharmacology; S2: predicting disease targets related to liver cancer ascites; S3: constructing an intersection target network of active ingredient targets and disease targets, and performing protein interaction network analysis; S4: performing GO function and KEGG pathway enrichment analysis on the intersection targets; S5: establishing a medicine, ingredient, disease, target, and pathway network; S6: verifying the binding activity of the core targets and active ingredients through molecular docking; S7: establishing a liver cancer ascites model through animal experiments to verify the efficacy; S8: analyzing the action mechanism of the medicine by integrating network pharmacology and animal experiment data.

4. The method according to claim 3, wherein the method is used for analyzing the mechanism of an external medicine for treating liver cancer ascites. In S1, the active ingredient screening adopts the TCMSP database, and the parameters are set as follows: molecular weight ≤500, number of hydrogen bond donors ≤5, number of hydrogen bond acceptors ≤10, octanol-water partition coefficient in -2-5, and number of rotatable bonds ≤10.

5. The method of claim 3, wherein the drug is selected from the group consisting of sorafenib, regorafenib, and lenvatinib.

5. The method of claim 3, wherein the drug is sorafenib. In S1, the target prediction adopts the SwissTargetPrediction database, and the probability threshold is ≥0.

1. The gene symbol is converted through the UniProt database.

6. The method of claim 3, wherein the mechanism of action of the external medicine for treating hepatocarcinoma ascites is analyzed. In S3, the intersection target network is constructed using the venny tool, the PPI network is constructed through the STRING database, the confidence is >0.7, and the topological analysis is performed using the Cytoscape software. The screening criteria include that the degree centrality, closeness centrality and betweenness centrality are all greater than the median.

7. The method according to claim 3, wherein the mechanism of action of the external medicine for treating hepatocarcinoma ascites is analyzed. In S7, the animal experiment adopts a Kunming mouse ascites model induced by H22 liver cancer cells. The groups include a blank group, a model group, a positive drug group and a medicine treatment group. The medicine treatment group includes a low-dose and a high-dose external patch treatment. 8.The method of claim 7, wherein the method is used for analyzing the mechanism of an external medicine for treating liver cancer ascites. In S7, the method for verifying the efficacy includes detecting the mouse abdominal circumference, ascites volume, serum ALT, AST and ALB levels, liver tissue pathological HE staining, and liver tissue IL-6, AKT1, ESR1 and JUN protein expression.

9. The method of claim 3, wherein the mechanism of action of the external medicine for treating hepatocarcinoma ascites is analyzed. The action mechanism is a multi-target and multi-pathway synergy, wherein the targets include IL-6, AKT1, ESR1 and JUN, and the pathways include cancer pathway, hepatitis B pathway and virus carcinogenic pathway.

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