CN113823364B - Method for analyzing mechanism of treating liver cancer based on network pharmacology and 1,2,3,4, 6-O-pentagalloylglucose - Google Patents

Method for analyzing mechanism of treating liver cancer based on network pharmacology and 1,2,3,4, 6-O-pentagalloylglucose Download PDF

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CN113823364B
CN113823364B CN202111173761.3A CN202111173761A CN113823364B CN 113823364 B CN113823364 B CN 113823364B CN 202111173761 A CN202111173761 A CN 202111173761A CN 113823364 B CN113823364 B CN 113823364B
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pentagalloylglucose
liver cancer
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protein
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CN113823364A (en
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王丽梅
王宏勋
易阳
侯温甫
闵婷
艾有伟
毕经会
雷秋琪
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Wuhan Polytechnic University
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Abstract

The invention discloses a method for analyzing a mechanism of pentagalloylglucose for treating liver cancer based on network pharmacology, which comprises the following steps: the Pubchem obtains the 2D structure of 1,2,3,4, 6-O-pentagalloylglucose and obtains a drug target; obtaining disease targets, screening 363 intersection targets of the drug and the disease, constructing a protein-protein interaction network by using a STRING database, inputting the protein interaction network into a Cytoscape to determine key target proteins, and then carrying out GO enrichment and KEGG enrichment channel analysis of the key target proteins to determine that 1,2,3,4, 6-O-pentagalloylglucose mainly plays a role through a P53 signal channel. On the basis of network pharmacology, a cell model is established to verify the therapeutic effect of the 1,2,3,4, 6-O-pentagalloylglucose on liver cancer.

Description

Method for analyzing mechanism of treating liver cancer based on network pharmacology and 1,2,3,4, 6-O-pentagalloylglucose
Technical Field
The invention belongs to the field of pharmacology, and particularly relates to a method for analyzing a mechanism of treating liver cancer based on network pharmacology, namely 1,2,3,4, 6-O-pentagalloylglucose.
Background
Cancer is an important obstacle to increasing life span worldwide, resulting in nearly 1000 tens of thousands of deaths in 2020. Among the many cancers, liver cancer is ranked fifth among the global morbidity and second among the mortality rates of men. 50% of new liver cancer cases worldwide occur in China, which poses a serious threat to public health. Early-diagnosis hepatocellular carcinoma patients have the opportunity to receive excision surgery, local destructive therapy or liver transplantation, and surgery recurrence or advanced liver cancer patients mostly adopt other systemic treatment methods such as chemotherapy, radiotherapy, targeted therapy and the like. At present, the targeting drugs for liver cancer have poor effect in the aspect of tumor treatment, so that the development of new targeting drugs has very important significance.
1,2,3,4, 6-O-pentagalloylglucose (1, 223, 446-penta-O-galloyl-beta-D-glucose, beta-PGG) is a polyphenol tannic acid monomer compound, and has various biological and pharmacological activities including antiviral, anti-inflammatory, antimicrobial, antidiabetic, etc. 1,2,3,4, 6-O-pentagalloylglucose has in vivo anticancer activity, and can inhibit various tumors such as colon cancer, breast cancer, prostatic cancer, lung pancreatic cancer, etc.
However, few reports on 1,2,3,4, 6-O-pentagalloylglucose for treating liver cancer exist at present.
Network pharmacology is a method of predicting the mechanism of action of drugs at the molecular level by means of databases. The network pharmacology can reveal complex relations between biological systems and medicines through information such as a molecular network, a network target point and the like which are extended by target molecules, and provides a new thought for the fields of discovery of bioactive substances, mechanism research, research and development of new medicines and the like. Many drugs in the past revealed their inherent association through network pharmacology. Therefore, there is a need in the art to explore the molecular mechanism of 1,2,3,4, 6-O-pentagalloylglucose for treating liver cancer using cellular experiments in combination with network pharmacological methods.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the method for analyzing the action mechanism of the 1,2,3,4, 6-O-pentagalloyl glucose for treating liver cancer is provided, the action mechanism of the 1,2,3,4, 6-O-pentagalloyl glucose for treating liver cancer is verified through network pharmacology and combination of in vitro experiments, the experimental result of in vitro pharmacodynamics corresponds to a key target obtained through network pharmacology and a related passage, and a potential molecular mechanism is determined, so that references are provided for subsequent development and application.
In order to achieve the above object, the present invention provides a method for treating liver cancer mechanism based on network pharmacology analysis of 1,2,3,4, 6-O-pentagalloylglucose, comprising the following steps, which are sequentially performed,
s1, obtaining an action target point of 1,2,3,4, 6-O-pentagalloylglucose medicine
And 2D structure of 1,2,3,4, 6-O-pentagalloyl glucose is obtained from a Pubchem database by taking 1,2,3,4,6-Pentakis-O-galloyl-beta-D-glucose as a keyword, and an sdf file of the drug structure is imported into a PharmMappe database and a SwissTargetPresection database to be combined and de-duplicated to obtain a drug related target.
S2, identification of disease action targets
Searching liver cancer related targets by taking a 'river cancer' as a keyword in a GeneCards database, and taking intersection of the obtained disease targets and the drug targeting genes obtained in the S1, wherein the obtained intersection genes are drug-disease intersection target genes.
S3, constructing a medicine-disease intersection target gene protein interaction diagram, and screening a core gene
Inputting the intersection target gene obtained in the step S2 into a String database, selecting a species of 'Homo sapiens', setting the lowest relation score to be 0.4, and removing scattered free proteins to obtain a protein interaction map. And (3) exporting the obtained protein interaction relation, inputting the exported protein interaction relation into software cytoscape3.7.1, and acquiring the eight top-ranked core genes by using a MCC calculation method in an plugin by using a cytoHubba plugin in the software.
S4, carrying out enrichment analysis on the core genes to analyze the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose for treating liver cancer
GO and KEGG analysis is performed by using an R packet clusterProfiler, enrichplot, P <0.05 is used as a screening condition, paths with the top ten and the top fifteen ranks are respectively screened out, and visualization is performed by using an R packet ggplot 2.
S5, construction of liver cancer in-vitro model
Human liver cancer (HepG 2) cells were cultured in an incubator at 37℃with 5% CO 2. The culture medium is supplemented with 10% inactivated fetal calf serum, 100U/ml penicillin and 10mg/L streptomycin. Sucking out the culture medium after the cells are attached, washing twice by PBS, and continuing to culture; when the cells grow to 80% -90%, digestion and passage are carried out by trypsin, and the passage is carried out once every 1-2 days; taking cells in logarithmic growth phase for later use.
S6, 1,2,3,4, 6-O-pentagalloylglucose inhibits proliferation and apoptosis of liver cancer cells and verification of related pathways
Preparing standard 1,2,3,4, 6-O-pentagalloylglucose into different concentrations, respectively treating liver cancer cells, and performing cell proliferation experiments, cell cycle detection, apoptosis detection, mitochondrial membrane potential detection and intracellular calcium ion concentration detection; in the aspect of channel verification, the method is divided into two layers, namely a gene layer, to carry out real-time fluorescence quantitative PCR, and a protein layer adopts a western blotting method.
According to a preferred embodiment of the present invention, the verification of the gene pathway in step S4 is mainly the P53 signal pathway obtained by GO and KEGG enrichment analysis.
According to a preferred embodiment of the present invention, the proliferation test of human liver cancer (HepG 2) cells in step S6 is performed by dissolving standard 1,2,3,4, 6-O-pentagalloylglucose in a small amount of dimethyl sub-maple, respectively, diluting the mixture to 0, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL with a culture medium, and adding the same concentration and volume of pentafluoroethane to a control group. Inoculating S5 logarithmic growth human liver cancer (HepG 2) cells into a 96-well plate, wherein each group of five compound wells; after 24 hours of incubation, the medium in the wells was aspirated, washed twice with PBS, incubated with equal amounts of 1,2,3,4, 6-O-pentagalloylglucose and pentafluoroeturacil (positive control) at different concentrations (0, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) for 24 and 48 hours, the well plates were thrown out of the drug-containing medium after the incubation time, washed with PBS, incubated in 10. Mu.L of CCK-8 solution per well in an incubator for three hours, and the well plates were placed in an ELISA reader to read absorbance at 450nm, and cell proliferation inhibition was calculated from the absorbance.
According to a preferred embodiment of the present invention, in the step 6, the cell cycle test is performed on human liver cancer (HepG 2) cells using a DNA content detection kit (purchased from beijing soleba technologies). Collection of HepG2 cells in logarithmic growth phase 1X 10 per well 6 Inoculating 1ml of culture medium in each hole of a six-hole plate, incubating for 24 hours in an incubator, and then adding different concentrations of drug-containing culture mediums to treat for 24 hours and 48 hours respectively; after the time, the cells were washed with pre-chilled PBS to adjust the cell concentration to 1X 10 6 Cells were collected by centrifugation at 1500 rpm for 5min per ml (supernatants were pooled and centrifuged together). After centrifugation, the supernatant was removed and the cells were fixed with 500. Mu.L of pre-chilled 70% ethanol for two hours overnight. Removing the fixing solution before dyeing, adding 100 mu L of RNaseA solution for resuspension, carrying out water bath at 37 ℃ for 30min, adding 400 mu L of PI dyeing solution, incubating at 4 ℃ for 30min in a dark place, and detecting by a flow cytometer.
According to a preferred embodiment of the present invention, the apoptosis test of the human liver cancer (HepG 2) cells in step 6 is performed by using BBcellProbe TM An Annexin V-FITC apoptosis assay kit (purchased from Shanghai Bei Bo Biotechnology Co.) was used for apoptosis detection. After cell culture for 24 hours, the cells are treated by adding the same amount of the drug-containing culture medium with different concentrations for 48 hours respectively. After completion of the treatment, cells were collected after centrifugation by digestion with EDTA-free pancreatin, washed twice with pre-chilled PBS, the PBS was discarded, and cells were suspended in 400. Mu.L of Annexin V-binding solution at a concentration of approximately1×10 6 5 mu LAnnexinV-FITC staining solution is added per ml, the mixture is gently mixed, and then the mixture is stained for 15min at 4 ℃ in a dark place, 10 mu LPI staining solution is added, and the mixture is gently mixed, and then shading incubation is carried out at 4 ℃ for 5min, and the detection is carried out by a flow cytometer.
According to a preferred embodiment of the present invention, the mitochondrial membrane potential test of the human liver cancer (HepG 2) cells in step 6 is performed using a mitochondrial membrane potential detection kit (JC-10) (available from beijing solebao technologies). Adding the same amount of the drug-containing culture medium containing different concentrations of drugs for 48 hours. Treatment with fluorescent lipophilic positive probe JC-1 (5, 5', 6' -tetra CHLORO-1,1', 3' -TETRAETHYL-IMIDACARBCYANINEIODIDE) following mitochondrial membrane potential kit protocol, flow cytometer detection
According to a preferred embodiment of the present invention, the human liver cancer (HepG 2) cells in step 6 are subjected to mitochondrial membrane potential assay using BBcellProbe TM F3 assay measures intracellular calcium ion concentration. Adding the same amount of the drug-containing culture medium containing different concentrations of drugs for 48 hours. Then 100. Mu.l of BBcellProbeTMF3 solution was added. After 20min of incubation, 1ml Hanks Balanced Salt Solution (HBSS) was added for 40min of incubation in the dark. The fluorescence intensity was measured by a microplate reader (Thermal Lab system, finland) at excitation/emission wavelengths of 488-506 nm.
According to a preferred embodiment of the present invention, the real-time fluorescent quantitative PCR assay of the human liver cancer (HepG 2) cells in step 6 is performed by extracting total RNA from the HepG2 cells using a total RNA extraction kit (omeg Co., USA) according to the gene shown in the P53 signal pathway map, and reverse transcribing the RNA into cDNA on ice using a reverse transcription kit (takara Co., japan). Beta-actin was used as an endogenous control, with 2, using a quantitative PCR system -ΔΔ-CT qRT-PCR was performed by calculating the relative fold change.
According to a preferred embodiment of the present invention, the western blotting experiment performed on the human liver cancer (HepG 2) cells in step 6 is: hepG2 cells were incubated with 1,2,3,4, 6-O-pentagalloylglucose for 48h, washed 2 times with 1mL of PBS, and transferred to a 1.5mL centrifuge tube. mu.L of cell lysate (10 mM Tris-C1 pH8.0,1mM EDTA,20%SDS,5mMDTT,10mM PMSF) was added and lysed on ice for 30min, sonicated 10 times for 2s each. Centrifugation was carried out at 12000rpm at 4℃for 5min, and the supernatant was collected and assayed for protein content using the BCA protein quantification kit. The protein sample was added with 1/4 of the sample volume of protein loading buffer, denatured at 95℃for 5min, cooled to room temperature and centrifuged at 12000rpm at 4℃for 5min. The protein samples were separated by electrophoresis in 10% SDS-polyacrylamide gel, cut after running out of the protein maker, and transferred to membrane under 100V, 90min, ice bath conditions. PVDF membranes were blocked with TBST (5% skim milk) at room temperature for 1h and incubated overnight at 4 ℃ with primary antibody. Washed three times with TBST for 5min each. Secondary antibody was added and incubated at room temperature for about 1h. The TBST was washed three times for 5min each. ECL luminescent substrate was added and scanned with a gel imager.
Through the design scheme, the invention has the following beneficial effects: a method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloyl glucose for treating liver cancer is characterized in that the action mechanism of 1,2,3,4, 6-O-pentagalloyl glucose for treating liver cancer is verified through network pharmacology and in vitro experiments, the experimental result of the in vitro pharmacodynamics is corresponding to key targets and related paths obtained through network pharmacology, potential molecular mechanisms of the key targets and the related paths are determined, and references are provided for subsequent development and application.
Further, pubchem acquires the 2D structure of 1,2,3,4, 6-O-pentagalloylglucose, and database PharmMapper, swissTargetPrediction acquires a drug target; obtaining disease targets by GeneCards, screening 363 intersection targets of the drug and the disease, constructing a protein-protein interaction network by STRING database, inputting the protein interaction network into a Cytoscape, determining key target proteins by using cytoHubba, enriching GO of the key target proteins by R package clusterProfile, enrichplot and analyzing KEGG enrichment channels, and determining that 1,2,3,4, 6-O-pentagalloylglucose mainly plays a role through P53 signal channels. On the basis of network pharmacology, a cell model is established to verify the therapeutic effect of the 1,2,3,4, 6-O-pentagalloylglucose on liver cancer. In vitro experiments show that the 1,2,3,4, 6-O-pentagalloylglucose plays an important role in proliferation of liver cancer cells, induction of apoptosis and cell cycle retardation. The real-time fluorescence quantification and enzyme linked immunosorbent assay are used for further elucidating that 1,2,3,4, 6-O-pentagalloylglucose induces apoptosis of cells by blocking a P53 signal pathway.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a network diagram of 1,2,3,4, 6-O-pentagalloylglucose-target-liver cancer obtained by network pharmacology in the method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose for treating liver cancer.
FIG. 2 is a network diagram of a key pharmacological target hub gene of a method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer.
FIG. 3 is a schematic diagram showing functional enrichment of network pharmacology GO in the method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer.
FIG. 4 is a schematic diagram showing the functional enrichment of network pharmacology KEGG according to the method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer.
FIG. 5 shows the effect of the in vitro pharmacodynamics experiment 1,2,3,4, 6-O-pentagalloylglucose and pentafluoroeturacil on inhibition of proliferation of human liver cancer (HepG 2) cells, according to the method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose on liver cancer.
FIG. 6 is an in vitro pharmacodynamic experiment of the effect of 1,2,3,4, 6-pentagalloyl-beta-D-glucose on HepG2 cell cycle for a method of analyzing the mechanism of action of 1,2,3,4, 6-O-pentagalloyl-glucose on liver cancer according to the present invention.
FIG. 7 shows the effect of 1,2,3,4, 6-pentagalloyl-beta-D-glucose on apoptosis of HepG2 in vitro pharmacodynamic experiments of a method for analyzing the mechanism of action of 1,2,3,4, 6-O-pentagalloyl-glucose on liver cancer.
FIG. 8 shows the effect of 1,2,3,4, 6-pentagalloyl-beta-D-glucose on mitochondrial membrane potential of HepG2 cells in vitro pharmacodynamics experiments of a method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloyl-glucose for treating liver cancer.
FIG. 9 shows the results of gene detection of HepG2 cells in vitro pharmacodynamics experiments, which are a method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer.
FIG. 10 shows the results of protein detection of HepG2 cells in vitro pharmacodynamics experiments, which are a method for analyzing the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer.
FIG. 11 shows the results of protein expression associated with HepG2 cells treated by 100. Mu.g/mL of 1,2,3,4, 6-O-pentagalloylglucose for 24 hours.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
Examples
This example is for illustrating a method for analyzing the mechanism of action of 1,2,3,4, 6-O-pentagalloylglucose in treating liver cancer, comprising a network pharmacological analysis, an in vitro pharmacodynamic analysis:
network pharmacological analysis
The structural formula of the sdf format of 1,2,3,4, 6-O-pentagalloylglucose was obtained from the PubCHem database, and potential binding genes for 1,2,3,4, 6-O-pentagalloylglucose were predicted using the PharmMapper (http:// lilab-mechanism/summitfile.html), swissTargetPresection (http:// www.swisstargetprediction.ch /) platform. Finally, 372 genes were predicted to be 1,2,3,4, 6-O-pentagalloylglucose binding genes. 16731 liver cancer-related genes were obtained from the GeneCard (https:// www.disgenet.org /) database. The drug gene and the disease gene are intersected to obtain 363 crossed genes, and the 1,2,3,4, 6-O-pentagalloylglucose can regulate the progress of liver cancer through the crossed genes. To further investigate the role of 1,2,3,4, 6-O-pentagalloylglucose in liver cancer, a "1,2,3,4, 6-O-pentagalloylglucose-target-liver cancer" network was constructed using cytoscape3.7.1 software, see figure 1.
And (3) introducing the predicted drug target and the GC related genes into a string database for PPI analysis, wherein the lowest relation score is set to be 0.4, and obtaining a protein interaction map. The resulting protein interactions were mapped into cytoscape3.7.1 for visual processing, and the central target proteins of front 8 in the network were found to be TP53, IGF1, EGFR, VEGFA, CASP3, MMP2, MMP9, SRC, respectively, using the MCC calculation method in cytoHubba plug-in, see fig. 2.
In order to elucidate the mechanism of action of 1,2,3,4, 6-O-pentagalloylglucose on liver cancer, GO and KEGG analyses were performed on 8 hub genes by R package. The GO database has three general categories, namely biological processes (Biological Process, BP), cellular components (Cellular Component, CC) and molecular functions (Molecular Function, MF), respectively, each describing the molecular functions that the gene product may perform, the cellular environment in which it is located, and the biological processes involved, and the GO function enrichment results indicate that the GO function enrichment results are mainly related to negative regulation of the apoptosis process, positive regulation of the peptide tyrosine phosphorylation, etc.; the KEGG results indicate that they are mainly related to the RAP1 signal path, the P53 signal path. By combining the analysis results of GO and KEGG, the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose on liver cancer is mainly related to apoptosis, and the path P53 signal path with the largest correlation with apoptosis is selected for further verification, as shown in figures 3 and 4.
In vitro pharmacodynamic analysis
Proliferation experiments of 1,2,3,4, 6-O-pentagalloylglucose in inhibiting liver cancer cells (HepG 2)
HepG2 cells were treated with 1,2,3,4, 6-O-pentagalloylglucose at various concentrations for 24 and 48 hours, respectively, and the viability of the cells was examined using CCK-8 reagent. As can be seen from Table 1, the IC50 value of the proliferation inhibition rate at 24 is 40.85 mug/mL which is slightly lower than that of the positive control group; the IC50 value at 48 hours was 28.50. Mu.g/mL, which is equivalent to that of the positive control group. The experiments show that 1,2,3,4, 6-O-pentagalloylglucose inhibits proliferation of liver cancer cells (HepG 2), as shown in FIG. 5.
TABLE 1 IC50 of 1,2,3,4, 6-O-pentagalloylglucose, 5-FU for HepG2
Influence of 1,2,3,4, 6-O-pentagalloylglucose on liver cancer cell cycle
The result of treating liver cancer cell cycle by detecting 1,2,3,4, 6-O-pentagalloylglucose by PI staining method is shown in the following graph. As shown in FIG. 6, it was found that 1,2,3,4, 6-O-pentagalloylglucose at a low concentration can cause liver cancer cells to remain in S phase, and 1,2,3,4, 6-O-pentagalloylglucose at a high concentration can cause liver cancer cells to remain in G0/G1 phase.
Influence of 1,2,3,4, 6-O-pentagalloylglucose on apoptosis of liver cancer cells
Detecting the change condition of apoptosis after treating liver cancer HepG2 cells by adopting an Annexin V-FITC/PI double-staining method, wherein the change condition is the change condition of apoptosis after treating liver cancer HepG2 cells by adopting 1,2,3,4, 6-O-pentagalloylglucose. The apoptosis ratio gradually increased after 48h of treatment of cells with 1,2,3,4, 6-O-pentagalloylglucose at different concentrations as shown in FIG. 7. The apoptosis rates of 100 mug/mL and 200 mug/mL are 36.44% and 51.02% respectively, which are significantly improved compared with the control group. The above results indicate that 1,2,3,4, 6-O-pentagalloylglucose can induce mitochondrial-dependent endogenous apoptosis in HepG2 cells.
Influence of 1,2,3,4, 6-O-pentagalloylglucose on membrane potential of liver cancer cell mitochondria
Mitochondrial membrane potential conditions of cancer cells were detected using mitochondrial membrane potential detection kit (JC-10). The membrane potential of liver cancer cells treated with the medicines is found to be reduced after the liver cancer cells are treated for 48 hours by 1,2,3,4, 6-O-pentagalloylglucose with different concentrations, and the mitochondrial membrane potential is reduced along with the change of the concentration, as shown in fig. 8, the mitochondrial membrane potential is reduced by 25.61% when the concentration reaches 200 mug/mL, which indicates that the induction of liver cancer cell apoptosis by 1,2,3,4, 6-O-pentagalloylglucose is accompanied by the reduction of the mitochondrial membrane potential and the increase of the permeability of the mitochondrial membrane potential.
Influence of 1,2,3,4, 6-O-pentagalloylglucose on the concentration of calcium ions in liver cancer cells
The change of the intracellular calcium ion concentration measured by the BBcellProbeTMF3 assay is shown in figure 9, the concentration of the intracellular free calcium ion is increased in a concentration-dependent manner after the cells are treated for 48 hours by using PGG with different concentrations, and the concentration of the intracellular calcium ion is increased by 78% when the concentration of the drug is 200 mug/mL, compared with a control group, the change of the intracellular calcium ion concentration is obvious, and the apoptosis of liver cancer cells induced by 1,2,3,4, 6-O-pentagalloylglucose has a close relationship with the increase of the intracellular free calcium ion concentration.
Influence of 1,2,3,4, 6-O-pentagalloylglucose on expression of liver cancer cell genes
The quantitative real-time PCR method was used to detect changes in intracellular apoptosis genes (P53, caspase-3, caspase-9, cytochrome c, bax, bcl-2, P21, cyclnD, IGF-BP3, PERP, PUMA) and the primers for the quantitative real-time PCR are shown in Table 2. HepG2 cells were treated with 100. Mu.g/mL 1,2,3,4, 6-O-pentagalloylglucose for 24h, and the related gene expression was shown in FIG. 10, which showed an increase and a very significant difference (P < 0.01) in the mRNA expression levels compared to P21, PUMA, PERP, IGF-BP3, caspase-9, cytochrome C in the control group cells; mRNA expression levels of Bax and P53 genes are increased and have significant differences (P < 0.05); the expression level of mRNA of the CyclinD gene is extremely obviously reduced (P is less than 0.01); the mRNA expression level of Bcl-2 gene was not significantly changed.
TABLE 2
Influence of 1,2,3,4, 6-O-pentagalloylglucose on expression of liver cancer cell proteins
The intracellular apoptosis genes (P53, caspase-3, caspase-9, cytochrome c, bax, bcl-2, P21, cyclinD, IGF-BP3, PERP, PUMA) were tested for changes using a western blot method. The HepG2 cells are treated by 100 mug/mL of 1,2,3,4, 6-O-pentagalloylglucose for 24 hours, the related protein expression is shown in figure 11, compared with a control group, the expression quantity of PUMA protein is increased and has obvious difference (P < 0.05), the Western blot detection result shows that the expression quantity of cytochrome C is increased and has obvious difference (P < 0.05), the caspase-3 protein is sheared and activated, and the expression quantity of the caspase-3 and caspase-9 proteins after activation is increased but has no obvious difference. In addition, the Western blot detects the change of the Bax and Bcl-2 protein content, and the result shows that the expression level of the Bax protein in the HepG2 cell is increased, the expression level of the Bcl-2 protein is reduced, and no obvious difference exists. The Western blot detects the change of the content of the P53 protein, and the result shows that the content of the P53 protein is increased and has extremely obvious difference (P is less than 0.01) in the process of inducing apoptosis of HepG2 cells by 1,2,3,4, 6-pentagalloyl-beta-D-glucose. Activated P53 regulates the expression of downstream P21 protein, resulting in increased P21 protein content in HepG2 cells with significant differences (P < 0.05).
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.

Claims (9)

1. A method for analyzing 1,2,3,4, 6-O-pentagalloylglucose based on network pharmacology to treat liver cancer mechanism, comprising the steps of:
s1, obtaining an action target point of 1,2,3,4, 6-O-pentagalloylglucose medicine
Obtaining a 2D structure of 1,2,3,4, 6-O-pentagalloyl glucose from a Pubchem database by taking 1,2,3,4,6-Pentakis-O-galloyl-beta-D-glucose as a keyword, and importing an sdf file of a drug structure into a PharmMappe database and a SwissTargetPresection database to be combined and de-duplicated to obtain a drug related target;
s2, identification of disease action targets
Searching a liver cancer related target in a GeneCards database by taking a 'river cancer' as a keyword, and carrying out intersection on the obtained disease target and the drug targeting gene obtained in the step S1, wherein the obtained intersection gene is a drug-disease intersection target gene;
s3, constructing a medicine-disease intersection target gene protein interaction diagram, and screening a core gene
Inputting the intersection target gene obtained in the step S2 into a String database, selecting a species of 'Homo sapiens', setting the lowest relation score to be 0.4, and removing scattered free proteins to obtain a protein interaction map; exporting the obtained protein interaction relation, inputting the exported protein interaction relation into software cytoscape3.7.1, and acquiring eight top-ranked core genes by using a cytoHubba plug-in the software and using an MCC calculation method in the plug-in;
s4, carrying out enrichment analysis on the core genes to analyze the action mechanism of 1,2,3,4, 6-O-pentagalloylglucose for treating liver cancer
Performing GO and KEGG analysis by using an R packet clusterProfiler, enrichplot, respectively screening paths ten and fifteen before ranking by taking P <0.05 as a screening condition, and performing visualization by using an R packet ggplot 2;
s5, construction of liver cancer in-vitro model
Taking human liver cancer cell HepG2, 37 ℃ and 5% CO 2 Is cultured in an incubator; the culture medium is supplemented with 10% inactivated fetal calf serum, 100U/ml penicillin and 10mg/L streptomycin; sucking out the culture medium after the cells are attached, washing twice by PBS, and continuing to culture; when the cells grow to 80% -90%, digestion and passage are carried out by trypsin, and the passage is carried out once every 1-2 days; taking cells in logarithmic growth phase for later use;
s6, 1,2,3,4, 6-O-pentagalloylglucose inhibits proliferation and apoptosis of liver cancer cells and verification of related pathways
Preparing standard 1,2,3,4, 6-O-pentagalloylglucose into different concentrations, respectively treating liver cancer cells, and performing cell proliferation experiments, cell cycle detection, apoptosis detection, mitochondrial membrane potential detection and intracellular calcium ion concentration detection; in the aspect of channel verification, the method is divided into two layers, namely a gene layer for real-time fluorescence quantitative PCR, and a protein layer adopts a western blotting method.
2. The method of claim 1, wherein the step S6 is performed by using P53 signaling pathway obtained by GO and KEGG enrichment analysis.
3. The method for treating liver cancer mechanism based on network pharmacology analysis 1,2,3,4, 6-O-pentagalloylglucose according to claim 1, wherein the proliferation experiment of the human liver cancer cells HepG2 in the step S6 is that the standard 1,2,3,4, 6-O-pentagalloylglucose is respectively dissolved in a small amount of dimethyl sub-maple, diluted into 0, 12.5 mug/mL, 25 mug/mL, 50 mug/mL, 100 mug/mL and 200 mug/mL by a culture medium, the same concentration and volume of pentafluoroethane is added into a control group, and the human liver cancer cells HepG2 growing in S5 logarithm are inoculated into a 96-well plate, wherein each group has five compound wells; sucking out the culture medium in the hole after culturing for 24 hours, washing twice by PBS, adding 1,2,3,4, 6-O-pentagalloylglucose with equal amounts of different concentrations and pentafluoroeturacil serving as a positive control group, incubating for 24 hours and 48 hours, throwing out the drug-containing culture medium of the pore plate after the time, adding 10 mu L of CCK-8 solution into each hole after washing by PBS, placing the pore plate in an incubator for incubation for three hours, placing the pore plate in an enzyme-labeled instrument, reading absorbance at 450nm, and calculating the cell proliferation inhibition rate according to the absorbance; the concentrations of the 1,2,3,4, 6-O-pentagalloylglucose with different concentrations are 0, 12.5 mug/mL, 25 mug/mL, 50 mug/mL, 100 mug/mL, 200 mug/mL.
4. The method for treating liver cancer mechanism based on network pharmacology analysis 1,2,3,4, 6-O-pentagalloylglucose of claim 1, wherein the cell cycle detection is performed using a DNA content detection kit; collection of HepG2 cells in logarithmic growth phase 1X 10 per well 6 Inoculating into six-hole plate, each hole1ml of culture medium, adding different concentrations of drug-containing culture medium after incubation for 24 hours in an incubator, and respectively treating for 24 hours and 48 hours; after the time, the cells were washed with pre-chilled PBS to adjust the cell concentration to 1X 10 6 Cells were collected by centrifugation at 1500 rpm for 5min, the supernatant was removed after centrifugation, and 500. Mu.L of pre-chilled 70% ethanol was added to the cells and fixed for two hours to overnight; removing the fixing solution before dyeing, adding 100 mu L of RNaseA solution for resuspension, carrying out water bath at 37 ℃ for 30min, adding 400 mu L of PI dyeing solution, incubating at 4 ℃ for 30min in a dark place, and detecting by a flow cytometer.
5. The method for treating liver cancer mechanism based on network pharmacology analysis 1,2,3,4, 6-O-pentagalloylglucose according to claim 1, wherein apoptosis detection is carried out by using a BBcellProbeTMAnnexin V-FITC apoptosis detection kit, respectively treating cells for 48 hours by adding equal amounts of drug-containing culture media with different concentrations after cell culture for 24 hours, collecting cells after digestion and centrifugation by using pancreatin without EDTA after treatment, washing the cells twice by precooling PBS, discarding PBS, suspending the cells with 400 mu L of Annexin V binding solution at a concentration of 1X 10E/ml, adding 5 mu L of Annexin V-FITC staining solution, gently mixing, shading for 15 minutes at 4 ℃, gently mixing, shading for 5 minutes at 4 ℃ after adding 10 mu L of PI staining solution, and detecting by a flow cytometer.
6. The method for treating liver cancer mechanism based on network pharmacology analysis 1,2,3,4, 6-O-pentagalloylglucose according to claim 1, wherein the method is characterized in that a mitochondrial membrane potential detection kit is used for adding an equal amount of a drug-containing medium containing drugs with different concentrations for 48 hours; the fluorescent lipophilic positive probe JC-1 was used, 5', 6' -TECHLORO-1, 1', 3' -TETRAETHYL-IMIDACARBOCYANINE IODIDE, treated following mitochondrial membrane potential kit instructions, and detected by flow cytometry.
7. The method for treating liver cancer mechanism based on network pharmacology analysis of 1,2,3,4, 6-O-pentagalloylglucose of claim 1, wherein the determination is performed by BBcellProbeTMF3 assayThe concentration of calcium ions in cells is treated for 48 hours by adding the same amount of medicine-containing culture medium containing medicines with different concentrations, and then 100 mu l of BBcelProbe is added TM F3 solution, after 20min of culture, adding 1ml of Hanks balanced salt solution HBSS, culturing in darkness for 40min, and measuring fluorescence intensity by an enzyme-labeled instrument under excitation/emission wavelength of 488-506 nm.
8. The method for treating liver cancer mechanism based on network pharmacology analysis of 1,2,3,4, 6-O-pentagalloylglucose according to claim 1, wherein qRT-PCR is performed by extracting total RNA from HepG2 cells using a total RNA extraction kit, reverse transcribing RNA into cDNA on ice using a reverse transcription kit, using a quantitative use system using β -actin as an endogenous control, and calculating relative fold change using an endogenous control method according to genes shown in a pathway diagram.
9. The method for treating liver cancer mechanism based on network pharmacology analysis of 1,2,3,4, 6-O-pentagalloylglucose of claim 1, wherein HepG2 cells are incubated with β -PGG for 48h, washed 2 times with 1mL PBS, and transferred to a 1.5mL centrifuge tube; adding 100 mu L of cell lysate, performing lysis on ice for 30min, performing ultrasound for 10 times for 2s each time, centrifuging at 12000rpm and 4 ℃ for 5min, collecting supernatant, measuring protein content by using BCA protein quantitative kit, adding 1/4 sample volume of protein loading buffer solution into protein sample, performing denaturation at 95 ℃ for 5min, cooling to room temperature, centrifuging at 12000rpm and 4 ℃ for 5min, performing electrophoretic separation on protein sample in 10% SDS-polyacrylamide gel, cutting gel after protein maker runs out, and transferring to membrane under the conditions of 100V, 90min and ice bath; the PVDF membrane was blocked with TBST for 1h at room temperature, incubated overnight at 4deg.C with primary antibody, washed three times with TBST for 5min each time, with secondary antibody added, incubated for about 1h at room temperature, washed three times with TBST for 5min each time, and scanned and analyzed by a gel imager with ECL luminescent substrate added.
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