Method for analyzing anti-bladder cancer action mechanism of paris polyphylla extract based on network pharmacology and experimental verification

Abstract

The invention discloses a method for analyzing an anti-bladder cancer action mechanism of a paris polyphylla extract on the basis of network pharmacology and experimental verification, which comprises the following steps: screening active components (polyphyllin I, polyphyllin II, polyphyllin V, polyphyllin VII, polyphyllin H and gracillin) of paris polyphylla through network pharmacology, and predicting action targets of the active components; an'active ingredient-target 'network is constructed, and KEGG enrichment analysis shows that the PI3K-Akt molecular marker can inhibit the progress of bladder cancer by regulating a PI3K-Akt signal channel. Furthermore, in-vitro experiment verification proves that the paris polyphylla extract can target GRB2 to inhibit a PI3K-Akt signal channel so as to play a role in resisting bladder cancer. The invention provides a scientific basis for the development of anti-bladder cancer medicines of paris polyphylla, and has the potential advantages of multiple targets and low toxicity.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a method for analyzing the mechanism of the anti-bladder cancer effect of a Paris polyphylla var. yunnanensis extract based on network pharmacology and experimental verification. BACKGROUND

[0002] Network pharmacology, as a new research method, can study the interaction between drugs and diseases from the overall level, and provides a powerful tool for revealing the mechanism of multi-component, multi-target and synergistic effect of traditional Chinese medicine. Bladder cancer is one of the most common malignant tumors of the urinary system, which seriously threatens human health. At present, the treatment methods for bladder cancer include surgery, chemotherapy, radiotherapy, etc., but there are problems such as high recurrence rate and large side effects, so it is of great significance to find new treatment drugs and methods. Paris polyphylla var. yunnanensis (Franch.) Hand.-Mzt. is a traditional Chinese medicinal material, which has various pharmacological activities. Previous studies have shown that the extract has an inhibitory effect on various tumor cells, but the specific mechanism of the anti-bladder cancer effect of the extract has not been fully elucidated. Paris polyphylla var. yunnanensis (Franch.) Hand.-Mzt. is a traditional Chinese medicinal material, which has various pharmacological activities. Previous studies have shown that the extract has an inhibitory effect on various tumor cells, but the specific mechanism of the anti-bladder cancer effect of the extract has not been fully elucidated. SUMMARY

[0003] The purpose of the present application is to further explore the mechanism of the anti-bladder cancer effect of the Paris polyphylla var. yunnanensis extract by combining network pharmacology and experimental verification, and to provide a theoretical basis and experimental basis for the development of new drugs for treating bladder cancer. The method of the present application is to screen active ingredients in the Paris polyphylla var. yunnanensis extract by network pharmacology, and to predict potential target points; to construct an active ingredient-target point interaction network, to screen key target points and signal pathways; to verify the binding ability of active ingredients and key target points by molecular docking; to detect the expression level of key target point proteins or genes by Western blot, to verify the prediction results of network pharmacology, and to obtain the mechanism of the anti-bladder cancer effect of the Paris polyphylla var. yunnanensis extract.

[0004] The active ingredients of the Paris polyphylla var. yunnanensis extract include Paris saponin I, Paris saponin II, Paris saponin V, Paris saponin VII, Paris saponin H and gracilistyl saponin.

[0005] The key target points include STAT3, IL2, PTAFR, PTPN1, PPM1B, PPP1CC, PPP2CA, PPP2R5A, HSD11B2, HSD11B1, F2, PTPRA, TYMS, FANCF, ATP1A1, GRB2, BCL2L1, TOP1, FGF1, FGF2, HPSE and VEGFA.

[0006] The key signal pathways include PI3K-Akt signal pathway, MAPK signal pathway and JAK-STAT signal pathway.

[0007] Compared with the prior art, the present application has the following advantages: The present application analyzes the potential mechanism of the extract of D. prainii against bladder cancer by a network pharmacology method, provides comprehensive target and signal pathway information for in-depth research, reveals that the extract of D. prainii may play an anti-bladder cancer role through multi-component, multi-target and multi-pathway synergistic action, and provides a new idea for the modernization of traditional Chinese medicine compounds. The network pharmacology prediction results are verified by cell experiments, which confirms that the extract of D. prainii has an inhibitory effect on the proliferation of bladder cancer cells, and the mechanism is closely related to the expression of PI3K-Akt pathway related proteins and core target proteins, thereby providing a reliable experimental basis for the development of new drugs for treating bladder cancer. The research method and results of the present application are helpful for in-depth understanding of the mechanism of traditional Chinese medicine, provide a scientific basis for the application of traditional Chinese medicine in the field of tumor treatment, and promote the development of the modernization of traditional Chinese medicine. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is a schematic diagram of the results of analyzing the main active ingredients in D. prainii by a high performance liquid chromatograph; Figure 2 is a schematic diagram of the results of analyzing the main active ingredients in D. prainii by UPLC-DAD-MS; Figure 3 is a schematic diagram of the results of analyzing the main active ingredients in D. prainii by UPLC-DAD-MS; Figure 4 is a schematic diagram of the results of analyzing the main active ingredients in D. prainii by UPLC-DAD-MS; Figure 5 is an active ingredient-target interaction network diagram; Figure 6 is a protein-protein interaction network result diagram; Figure 7 is a KEGG pathway enrichment analysis result; Figure 8 is a result of molecular docking of active ingredients PPI, PPII of D. prainii and GRB2; Figure 9 is a result of molecular docking of active ingredients PPV, PPVII of D. prainii and GRB2; Figure 10 is a result of molecular docking of active ingredients PPH, PPG of D. prainii and GRB2; Figure 11 is a T24 cell experiment result of PPT on the PI3K-Akt pathway, the upper graph is a protein electrophoresis diagram, and the lower graph is an expression amount statistical diagram; Figure 12Figure 1 is the experimental result of PPT on PI3K-Akt pathway of UM-UC-3 cells, the upper graph is a Western blotting graph, and the lower graph is a statistical result graph; Figure 13 Figure 2 is the experimental result of PPT on GBR2 siRNA, the upper graph is a Western blotting graph, and the lower graph is a statistical result graph; Figure 14 Figure 3 is the experimental result of PPT targeting GRB2 to inhibit PI3K-Akt pathway, the upper graph is a Western blotting graph, and the lower graph is a statistical result graph. DETAILED DESCRIPTION

[0009] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments, but these embodiments shall not be used to explain the limitation of the present application. In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the experimental materials are all commercially available unless otherwise specified. After reading the description of the present application, those skilled in the art can make various changes, modifications and modifications of the equivalents, which all fall within the scope defined by the claims of the present application.

[0010] In the examples, the dried rhizomes of D. yunnanensis Franch. Paris polyphylla were purchased from Kunming medicinal material market in Yunnan, and were identified as authentic products.

[0011] Reagents: RPMI 1640 medium (Gibco, USA), fetal bovine serum (Guangzhou Sike Biotechnology Co., Ltd.), trypsin (Gibco, USA), hydroxypropyl-β-cyclodextrin (Aladdin, Shanghai, China), Opti-MEM medium (Gibco, USA), Lipofectamine 2000 (Invitrogen, USA), h-GRB2 siRNA nucleic acid (Guangzhou Jidan Biotechnology Co., Ltd.). β

[0012] Cell lines: bladder cancer cells T24 and UM-UC-3 were purchased from Guangzhou Sike Biotechnology Co., Ltd.

[0013] Example 1: Screening of active ingredients of D. yunnanensis Franch. 1. The dried rhizomes of D. yunnanensis Franch. were crushed and passed through a 60-mesh sieve to obtain coarse powder; 2. According to the ratio of material to liquid of 1:10 (g:mL), 2% hydroxypropyl-β-cyclodextrin solution was added to the coarse powder of D. yunnanensis Franch., and after mixing, ultrasonic extraction (power 180W) was performed at 50°C for 30 minutes, followed by solid-liquid separation, and the solid was added with hydroxypropyl-β-cyclodextrin solution again; β β ​​- The cyclodextrin solution was extracted twice, and the extracts were collected and combined. The extracts were passed through a macroporous resin HPD600 column and eluted with 70% ethanol solution. The eluent was concentrated at 40°C and freeze-dried to obtain the Paris polyphylla extract (PPT). 3. Identification of hydroxypropyl groups in Paris polyphylla using UPLC-DAD-MS technique. β - The main active ingredient in the cyclodextrin extract. Chromatographic conditions: mobile phase: acetonitrile (A) and water (B); gradient elution program: 0-22 min, 35%-53% acetonitrile; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 35℃; detection wavelength: 203 nm. Mass spectrometry conditions: ESI negative ion mode (scanning range 50–3000 m / z); sheath gas flow rate: 10.0 L / min; sheath gas temperature: 350℃; drying gas temperature: 350℃; drying gas flow rate: 10.0 L / min; capillary voltage: 3500 V; nebulization pressure: 30 psi. See results Figures 1-4 As can be seen from the figure, UPLC-DAD-MS technology was used to identify the hydroxypropyl group of Paris polyphylla. β - The main active components in the cyclodextrin extract are: peak 1 is Paris polyphylla saponin VII (PPVII), peak 2 is Paris polyphylla saponin H (PPH), peak 3 is Paris polyphylla saponin II (PPII), peak 4 is diosgenin (PPG), peak 5 is Paris polyphylla saponin I (PPI), and peak 6 is Paris polyphylla saponin V (PPV).

[0014] Example 2: Prediction of potential targets and pathways of the main active ingredients of Paris polyphylla yunnanensis 1. The six active ingredients of Paris polyphylla selected from screening—Parisin I, Parisin II, Parisin V, Parisin VII, Parisin H, and Dioscorea cirrhosa—were sequentially searched in the PubChem database to obtain SDF format files of the SMILES and 2D structures of the ingredients. The obtained SDF format files of the SMILES or 2D structures were then imported into the Swiss TargetPrediction database to predict their potential targets. 2. Import the potential targets of the six active ingredients of Paris polyphylla into the STRING database, set the object as (homosapiens) to construct a protein-protein interaction network, set the confidence score ≥0.4, export the protein-protein interaction network data, and use Cytoscape 3.10.0 software for visualization analysis. Select "networkAnalyzer" to obtain the network topology parameters, and screen the core targets according to the Degree value. 3. Using the DAVID database, pathway enrichment analysis of six potential targets of active ingredients of Paris polyphylla was performed to identify the main signaling pathways involved. See results Figures 5-7 ,from Figure 5 As can be seen from the target prediction of 6 active ingredients of Paris polyphylla, 22 potential targets were identified, namely STAT3, IL2, PTAFR, PTPN1, PPM1B, PPP1CC, PPP2CA, PPP2R5A, HSD11B2, HSD11B1, F2, PTPRA, TYMS, FANCF, ATP1A1, GRB2, BCL2L1, TOP1, FGF1, FGF2, HPSE, and VEGFA; Figure 6 Protein-protein interaction network analysis further identified GRB2 as a key pivotal target; Figure 7 KEGG pathway enrichment analysis showed that the PI3K-Akt pathway was the most significantly enriched pathway.

[0015] 4. Download the crystal structure of the target protein (GRB2) from the PDB database and the ligand structure from PubChem for molecular docking verification; use AutoDock software to process the target protein and ligand, verify the binding energy between the active ingredient and the core target GRB2, and confirm their interaction. See results Figures 8-10 As can be seen from the figure, the binding energy range of Paris polyphylla saponin I, Paris polyphylla saponin II, Paris polyphylla saponin V, Paris polyphylla saponin VII, Paris polyphylla saponin H, diosgenin and GRB2 is -8.5 to -9.5, indicating that GRB2 binds well with the six active ingredients of Paris polyphylla.

[0016] Example 3: Cellular experiments to validate the results of network pharmacology predictions 1. Western blot assay T24 and UM-UC-3 bladder cancer cells in logarithmic growth phase were evenly seeded into 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, the cells were treated with different concentrations (7.5 μg / mL, 15 μg / mL, and 30 μg / mL) of PPT for 24 hours. Total protein was then extracted from the cells, and Western blotting was performed to detect p-PI3K, p-Akt, p-mTOR, and p-GSK3. β The expression level of p21 protein.

[0017] See results Figure 11 , 12 Western blot results showed that the key proteins of the PI3K-Akt pathway, p-PI3K, p-Akt, p-mTOR, and p-GSK3, were involved.β The increased levels of p21 protein and the decreased levels of p21 protein suggest that PPT can inhibit the PI3K-Akt pathway.

[0018] 2. siRNA experiment T24 and UM-UC-3 bladder cancer cells in logarithmic growth phase were evenly seeded into 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, transfection was performed. The siRNA-Lipid complex was prepared as follows: Tube A: 125 µL Opti-MEM + 5 µL Lipofectamine 2000 (gently mixed, incubated at room temperature for 5 min); Tube B: 125 µL Opti-MEM + 50 nM siRNA (final concentration, e.g., 5 µL of 10 µM siRNA). Tubes A and B were mixed (total volume 250 µL), gently mixed, and incubated at room temperature for 15-20 min. Then, 750 µL Opti-MEM was added and gently mixed. The old culture medium was removed, and the siRNA-liposome complex was added dropwise to the wells, gently shaken to mix, and after 6 hours, the medium was replaced with complete medium and cultured for another 36 hours. After h, total cell protein was extracted and subjected to Western blotting to detect knockdown efficiency and the levels of p-PI3K, p-Akt, p-mTOR, and p-GSK3. β The expression level of p21 protein.

[0019] See results Figure 13 , 14 As shown in the figure, the most effective siRNA (seq 2) enhanced PPT-induced inhibition of the PI3K-Akt pathway; mechanistic studies revealed that GRB2 knockdown significantly downregulated p-PI3K, p-Akt, p-mTOR, and p-GSK3. β Simultaneously, p21 was upregulated, reflecting that PPT targets GRB2 to inhibit the PI3K-Akt pathway.

[0020] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for analyzing the anti-bladder cancer mechanism of Paris polyphylla extract based on network pharmacology and experimental verification, characterized in that, Includes the following steps: The active ingredients in Paris polyphylla extract were screened using network pharmacology, and their potential targets were predicted. Construct an active ingredient-target interaction network to screen key targets and signaling pathways; The binding ability of active ingredients to key targets was verified through molecular docking. Western blot was used to detect the expression levels of key target proteins or genes, and the network pharmacology prediction results were verified to analyze the mechanism of action of Paris polyphylla extract against bladder cancer.

2. The method according to claim 1, characterized in that: The active ingredients in the extract of Paris polyphylla include Paris saponin I, Paris saponin II, Paris saponin V, Paris saponin VII, Paris saponin H, and diosgenin.

3. The method according to claim 1, characterized in that: Key targets include STAT3, IL2, PTAFR, PTPN1, PPM1B, PPP1CC, PPP2CA, PPP2R5A, HSD11B2, HSD11B1, F2, PTPRA, TYMS, FANCF, ATP1A1, GRB2, BCL2L1, TOP1, FGF1, FGF2, HPSE, and VEGFA.

4. The method according to claim 1, characterized in that: Key signal pathways include the PI3K-Akt signal pathway, the MAPK signal pathway, and the JAK-STAT signal pathway.

5. The method according to claim 1, characterized in that: In vitro cell experiments were performed using human bladder cancer cell lines T24 and UM-UC-3.

6. The method according to claim 1, characterized in that: Extracts from Paris polyphylla (Yunnan) inhibit the proliferation of bladder cancer cells by regulating the PI3K-Akt signaling pathway.

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