Application of periplocin in anti-hepatocellular carcinoma medicine and target screening method

Abstract

The invention discloses application of periplocin in anti-hepatocellular carcinoma drugs and a target screening method, and in-vitro experiment discussion verification is adopted. Through network pharmacology, a TCMSP database is used for screening key bioactive components of cortex periplocae, hepatocellular carcinoma related targets are screened in combination with transcriptomics sequencing data, an intersection is taken, and a network is constructed to screen out an FOXO1 key target. The cell activity and cell proliferation are detected through an MTT method and a clone formation experiment, cell cycle apoptosis is detected through flow cytometry, and it is further determined that the FOXO1 is low in expression in hepatocellular carcinoma tissue and is related to the survival time of a patient. The cortex periplocae component is analyzed, network pharmacology and cell experiments are combined to verify the anti-hepatocellular carcinoma active component and possible action mechanism of periplocin, a scientific basis is expected to be provided for fundamental research of anti-hepatocellular carcinoma pharmacodynamic substances of periplocin, and a novel hepatocellular carcinoma targeted therapeutic drug is found from the scientific basis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and more particularly to the application of periplocin in anti-hepatocellular carcinoma drugs and a method for screening targets. Background Art

[0002] Hepatocellular carcinoma (HCC) is a major global public health issue, ranking sixth in incidence and third in mortality from malignant tumors worldwide. With a five-year survival rate of 18%, HCC is the second most lethal cancer after pancreatic cancer. HCC accounts for approximately 75%-85% of all cases. In my country, less than 30% of HCC patients are detected and diagnosed early, and the average survival for patients with advanced HCC is less than one year. Furthermore, due to its insidious onset, HCC is often diagnosed at an advanced stage, and less than 30% of patients are eligible for radical surgery. Treatment options for HCC are diverse, including liver transplantation, hepatectomy, radiofrequency ablation, radiotherapy, and systemic therapy. However, these treatments have limited efficacy, with only a small percentage of patients achieving sustained clinical benefit. Furthermore, the heterogeneity and distant metastasis of HCC contribute to the high mortality rate, making HCC a highly lethal disease. Therefore, in-depth exploration of the molecular mechanisms underlying HCC pathogenesis and the identification of novel therapeutic targets and drugs are of great scientific and clinical significance.

[0003] Traditional Chinese medicine and natural medicine are valuable and abundant medical resources in China. In particular, Chinese herbal medicines have the advantages of high safety, diverse structures, good activity, and abundant natural resources. This has made the search for anti-tumor active ingredients from traditional Chinese medicine and natural products an important approach to anti-tumor drug research and development in recent years. Periploca sepium Bge, also known as Acanthopanax septoriae and Periploca sepium bark, is the dried root bark of the Asclepiadaceae plant. It has the effects of dispelling rheumatism, strengthening bones and muscles, and promoting diuresis and reducing swelling. Studies have shown that Periploca sepium Bge plays a role in the treatment of various malignancies, including nasopharyngeal carcinoma, esophageal cancer, and liposarcoma. Cardiac glycosides, the main component of Periploca sepium extracts, are believed to be the most important component in its anti-tumor mechanism. Perioplogenin (Per), the main active substance in Periploca sepium, is a naturally occurring cardiac glycoside compound with cardiotonic, anti-tumor, anti-inflammatory, and immunomodulatory effects. Studies have shown that periplocin can induce programmed necrosis in HaCaT cells through oxidative stress and improve skin lesions in TPA- and IMQ-induced psoriasis-like mouse models. Other studies suggest that periplocin can trigger apoptosis in colorectal cancer cells by activating the ROS-ER stress pathway through the BIP-eIF2a-CHOP and IRE1aASK1-JNK signaling pathways. Therefore, studying the anti-tumor mechanisms and targets of periplocin is of great significance for the development of anti-tumor drugs derived from Periploca odorata. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides an application of periplocin in anti-hepatocellular carcinoma drugs and a method for screening targets thereof, thereby providing more and better options for the treatment of cancer, especially hepatocellular carcinoma.

[0005] To achieve the above object, the present invention provides the following technical solution: application of periplocin in anti-hepatocellular carcinoma drugs, wherein the drugs include active ingredients and pharmaceutical excipients; the active ingredients contain periplocin.

[0006] The present invention is further provided that the medicine is used to treat primary hepatocellular carcinoma.

[0007] The present invention is further provided that the medicine is used to treat hepatocellular carcinoma.

[0008] The present invention is further configured as follows: the drug is used to inhibit the proliferation of hepatocellular carcinoma cells, reduce the migration and metastasis of hepatocellular carcinoma cells, and induce apoptosis of hepatocellular carcinoma cells.

[0009] The present invention is further configured as follows: the hepatocellular carcinoma cells are HCCLM3, HUH7 or SMMC-7721 cells.

[0010] The present invention also provides a method for screening targets of periplocin and hepatocellular carcinoma, comprising:

[0011] A. Database-based active ingredient screening and target prediction;

[0012] B. Construction of active ingredient-target network and screening of key active ingredients;

[0013] C. Regulatory network construction and key target screening;

[0014] D. Transcriptomic analysis and key target identification.

[0015] In summary, the present invention has the following beneficial effects:

[0016] The present invention provides a new anticancer use of periplocin, which has a good anticancer effect on hepatocellular carcinoma, especially hepatocellular carcinoma.

[0017] This invention identifies the key target FOXO1 through network pharmacology and transcriptomics studies. FOXO1 is lowly expressed in hepatocellular carcinoma tissues and is associated with short patient survival. This provides a basis and potential biomarkers for understanding the anti-hepatocellular carcinoma mechanism of periplocin, developing precise treatment strategies, and evaluating the prognosis of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1a 、 Figure 1b 、 Figure 1c and Figure 1d This is the network pharmacology screening result in Example 1 of the present invention.

[0019] Figure 2 This is the result that periplocin in Example 2 of the present invention can inhibit the proliferation of hepatocellular carcinoma cells.

[0020] Figure 3 This is the result that periplocin can inhibit the migration of hepatocellular carcinoma cells in Example 3 of the present invention.

[0021] Figure 4 This is the effect of periplocin in Example 4 of the present invention on apoptosis of hepatocellular carcinoma cells.

[0022] Figure 5a 、 Figure 5b and Figure 5c In Example 5 of the present invention, network pharmacology and transcriptomics were combined to screen the key targets of periplocin for anti-hepatocellular carcinoma. Periplocin can exert its anti-hepatocellular carcinoma effect by targeting FOXO1. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to the accompanying drawings.

[0024] Network pharmacology: All components of Cortex Periplocae were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. Pharmacokinetic (ADME) parameters were used to screen the components, with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, to identify key bioactive components of Cortex Periplocae. The TCMSP target prediction model was used to predict the targets of these active components, excluding duplicate targets to obtain a total of predicted targets. Hepatocellular carcinoma-related targets were screened using five databases: GeneCard, OMIM, PharmGkb, TTD, and DrugBank. These targets were then intersected with the TCMSP predicted targets for Cortex Periplocae active components to identify common targets. Using the network visualization software Cytoscape 3.7.2, we imported the identified key active ingredients and their targets from Cortex Periplocae into the network to construct an "active ingredient-target network" for Cortex Periplocae. We evaluated the network centrality using three topological metrics: betweenness centrality (BC), closeness centrality (CC), and degree centrality (DC). We then screened the key active ingredients in the "Cortex Periplocae active ingredient-target" network and selected periplogenin as the target for our next experimental validation.

[0025] Transcriptomics: Total RNA was extracted and detected from the control group and the medium-concentration group of periwinkle aglycone. Libraries were constructed and transcriptome RNA sequencing (RNA-seq) was performed to screen for differentially expressed mRNAs and conduct KEGG analysis. Aging-related targets were screened from the Geneage and GeneCard databases and intersected with the sequencing data. The network visualization software Cytoscape 3.7.2 was used to import the identified common targets and construct a "target network." The node network centrality was evaluated based on three topological characteristic indicators: betweenness centrality (BC), closeness centrality (CC), and degree of freedom (DC), to screen for key active ingredients in the network.

[0026] MTT: MTT assay was used to detect the survival rate of human hepatocellular carcinoma cells. HCCLM3, HUH7, and SMMC-7721 cells (1×10 4 Cells (cells / well) were seeded in 96-well plates and treated appropriately with different culture media. After 48 hours, MTT solution was added at a dose of 20 μL / well and incubated at approximately 37°C in a CO2 incubator for 4 hours. Formazan crystals were dissolved with 150 μL of dimethyl sulfoxide, and the optical density (OD) of the MTT solution was recorded using a microplate reader (absorbance at 490 nm). Cell viability and IC50 values ​​were calculated using GraphPad Pro Prism 9.0.

[0027] Colony formation assay: Human hepatocellular carcinoma cell lines HCCLM3, HUH7, and SMMC-7721 (1000 cells / well) were seeded in six-well plates and incubated overnight at 37°C in 5% CO2. Dimethyl sulfoxide (control) and periplocin (0.5, 1, and 2 μM) were added to the cells for 48 hours. The medium was replaced with fresh medium every two days to maintain cell growth for 14 days. Colonies were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed three times with PBS, and stained with crystal violet for 10 minutes.

[0028] EdU Experiment: EdU Staining with BeyoClick to Study Cell Proliferation TM EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, China, No. C0075S) was used. Cells were first washed three times with PBS for five minutes each. A predetermined amount of EdU was added to fresh culture medium. Cells were incubated in a 37°C CO2 incubator for two hours. Following incubation, cells were washed again with PBS twice for 10 minutes each. Cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes and then stained with Hoechst 33342 for 10 minutes. After washing again with PBS, cells were observed under an inverted microscope.

[0029] TRANSWELL experiment: According to the manufacturer's instructions, transwell filters (NEST, China, No. 725301) were used to observe cell migration. The cell density was 1×10 3 Serum-free medium was added to the upper chamber. Medium containing 10% FBS was added to the lower chamber. After 48 hours of drug treatment, cells were fixed with 4% paraformaldehyde, and unbroken cells were removed from the upper surface of the filter. Cells on the lower surface of the membrane were stained with 1% crystal violet for 15 minutes at room temperature in the dark. Migrated cells were recorded and counted under a light microscope. Images were captured under an inverted phase-contrast microscope.

[0030] Scratch test: cells (5×105 Cells (100 cells / well) were seeded in 6-well plates and allowed to adhere overnight. At 80–90% confluence, a "reference line" was drawn on the bottom of the plate using a sterile 10 μL pipette tip. After washing three times with phosphate-buffered saline (PBS), cells were further incubated with inhibitors or a control (DMSO) in culture medium to examine cell migration in the absence of cell growth. Micrographs of cells migrating across the reference line were taken at different areas using an inverted phase-contrast microscope at 0, 24, 48, and 72 hours after treatment. Migration rate was quantified by the distance cells migrated from the reference line toward the center compared to the control group.

[0031] Apoptosis assay: HUH7, HCCLM3, and SMMC-7721 cells were seeded in six-well culture plates and grown to 80% confluence in complete medium. The cells were then treated with dimethyl sulfoxide (control) and periwinklein (0.5, 1, and 2 μM) for 48 hours to assess the effects of these compounds on cell apoptosis. The cells were collected, washed twice in ice-cold PBS, and then resuspended in binding buffer according to the instructions of the apoptosis kit. The treated cells (as described above) were incubated simultaneously with fluorescein-labeled annexin V and PI. Annexin V binding buffer was then added to the mixture, and fluorescence was measured on a FACSCalibur (BD Biosciences, Baltimore, MD, USA). The data were analyzed using FlowJo software.

[0032] Example 1

[0033] This example uses network pharmacology to obtain the active ingredient of Periploca radix and targets the FOXO1 pathway to fight hepatocellular carcinoma. Figure 1a AB in), to obtain the key active ingredient of Periploca radiata ( Figure 1b C in the figure). Using five databases, GeneCard, OMIM, PharmGkb, TTD, and DrugBank, we screened hepatocellular carcinoma-related targets and obtained a total of 13,578 targets ( Figure 1b D in the figure, and intersected it with the predicted targets of TCMSP for the active ingredients of Periploca rotunda, and 27 targets were obtained ( Figure 1c The key active ingredients and their targets of Periploca rotunda were imported into the network visualization software Cytoscape 3.7.2 to construct the “active ingredient-target network” of Periploca rotunda ( Figure 1c Through network topology analysis, 8 key targets were found: FOXO1, PTGS1, HSP90AA1, PTGS2, PRKCA, BCL2, NR3C1, PGR ( Figure 1d G in ).

[0034] Example 2

[0035] This example shows that periplocin has a dose-dependent and time-dependent cytotoxic and anti-proliferative effect on HCC cells through MTT test, colony formation test and EDU test. Cell clone formation experiment shows that periplocin can inhibit the colony formation of human hepatocellular carcinoma cells in a concentration-dependent manner ( Figure 2 A in the figure). The MTT assay was used to detect the growth inhibition and cytotoxic effects of periplocin on HCC cells. The IC50 values ​​of the three cell lines were: HCCLM3: 1.1±0.6μM, HUH7: 1.587±0.17μM, SMMC-7721: 0.61±0.19μM ( Figure 2 B in the figure). EDU cell proliferation assay showed that periplocin could inhibit HCC cell proliferation in a dose-dependent manner ( Figure 2 These results indicate that periplocin can inhibit the proliferation of HCC cells.

[0036] Example 3

[0037] This example further observed the effect of periplocin on the migration ability of hepatocellular carcinoma cells. The scratch healing assay of HUH7 and HCCLM3 cells suggested that periplocin could inhibit the scratch healing ability of HCC cells ( Figure 3 In the Transwell migration experiment, the addition of drugs significantly reduced the migration ability of HCCLM3, HUH7, and SMMC-7721 cells ( Figure 3 B) in.

[0038] Example 4

[0039] This example further analyzed the effect of periwinkle aglycone on apoptosis of hepatocellular carcinoma cells using flow cytometry. Annexin V-FITC was used as a marker of cell apoptosis, and propidium iodide (PI) was used as a marker of cell viability. The results showed that periwinkle aglycone could promote the increase of apoptosis in HCCLM3, HUH7, and SMMC-7721 cells ( Figure 4 ).

[0040] Example 5

[0041] In this example, 1725 differentially expressed mRNAs were screened by transcriptomics, of which 1061 genes were upregulated and 664 genes were downregulated ( Figure 5a A in the figure) and KEGG signaling pathway enrichment analysis was performed on the differentially expressed genes, which included MAPK signaling pathway, cell aging-related pathway, cytomegalovirus infection, FoxO signaling pathway, etc. ( Figure 5aB in the figure). 75 common targets were obtained from the aging targets obtained from the Geneage and GeneCard databases and the differentially expressed genes after the action of periplocin ( Figure 5b C in). Import the common target into string (https: / / string-db.org) to obtain the TSV graph of PPI ( Figure 5b The obtained TSV graph was imported into the network visualization software Cytoscape3.7.2, and the node network centrality was first screened and evaluated using the three topological characteristic indicators BC, CC, and DC to screen out the key targets in the network ( Figure 5c E), and then screened again to obtain the final 11 key targets: EP300, FOXO1, HSP90AA1, BCL2, EGF, STAT3, SIRT1, TNF, JUN, BRCA1, TP53 ( Figure 5c Combined with previous studies ( Figure 1c Finally, FOXO1 was selected as the target. Molecular docking of FOXO1 and periplocin was performed, and computer molecular simulation docking was performed through Molecule Docking experiment. The results showed that periplocin formed a hydrogen bond with amino acid Asn240 of FOXO1 ( Figure 5c G in ), and the range of action is Binding energy: -4.122kcal / mol. This suggests that periwinkle aglycone may be a FOXO1 targeting agent. Based on the above results, this project analyzed the expression differences of FOXO1 mRNA in hepatocellular carcinoma tissues in the TCGA database. The results showed that FOXO1 mRNA expression was downregulated in hepatocellular carcinoma tissues (P = 1.256e-15) ( Figure 5c The H in the figure). The role of FOXO1 expression in the prognosis of patients with hepatocellular carcinoma was also explored. The Kaplan-Meier survival curve was drawn, and the results showed that the overall survival of patients with hepatocellular carcinoma with low FOXO1 expression was shorter, and the difference was statistically significant (P=0.018) ( Figure 5c The above results suggest that FOXO1 may become a biomarker with good prognosis for hepatocellular carcinoma.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the design concept of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of periplocoside in anti-hepatocellular carcinoma drugs, characterized in that: The medicine comprises active components and pharmaceutical excipients; the active components contain periplocin.

2. The use of periplocin in anti-hepatocellular carcinoma drugs according to claim 1, characterized in that: The medicine is used for treating primary hepatocellular carcinoma.

3. The use of periplocin in anti-hepatocellular carcinoma drugs according to claim 1, characterized in that: The medicine is used for treating hepatocellular carcinoma.

4. The use of periplocin in anti-hepatocellular carcinoma drugs according to claim 1, characterized in that: The drug is used for inhibiting the proliferation of hepatocellular carcinoma cells, inhibiting the migration of hepatocellular carcinoma cells, and inducing apoptosis of hepatocellular carcinoma cells.

5. The use of periplocin in anti-hepatocellular carcinoma drugs according to claim 4, characterized in that: The hepatocellular carcinoma cells are HCCLM3, HUH7 or SMMC-7721 cells.

6. A method for screening targets of periplococephalus aglycone and hepatocellular carcinoma, characterized in that: The steps include: A. Screening of active drug ingredients and target prediction based on TCMSP database; B. Construction of the active ingredient-target network of periplocoside aglycone and screening of key active ingredients; C. Construction of regulatory network and screening of key targets; D. Transcriptomic analysis and identification of key targets.

7. The method for screening targets of periplocin and hepatocellular carcinoma according to claim 6, characterized in that: In the active ingredient screening and target prediction based on the TCMSP database, all ingredients of Periploca serrata are retrieved through the database TCMSP, oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18 are used as screening conditions, and the key active ingredients of Periploca serrata are screened out according to the pharmacokinetic (ADME) parameters of each ingredient; the TCMSP target prediction model is used to predict the action targets of the above active ingredients, duplicate targets are excluded, and predicted targets are obtained.

8. The method for screening targets of periplocin and hepatocellular carcinoma according to claim 7, characterized in that: The construction of the active ingredient-target network of Perioplogenin and the screening of key active ingredients include using five databases, namely GeneCard, OMIM, PharmGkb, TTD and DrugBank, to screen hepatocellular carcinoma-related targets, and taking the intersection with the predicted targets of the active ingredients of Cortex Periplosum in the TCMSP database to obtain common targets; using Cytoscape3.7.2, the screened key active ingredients of Cortex Periplosum and their targets are imported into it to construct the "active ingredient-target network" of Cortex Periplosum; the centrality of the node network is evaluated according to three topological characteristic indicators, namely node betweenness centrality (BC), node compactness (CC) and node degree of freedom (DC), to screen the key active ingredients in the "Cortex Periplosum active ingredient-target" network, and selecting Perioplogenin as the object of experimental verification.

9. The method for screening targets of periplocin and hepatocellular carcinoma according to claim 8, characterized in that: In the regulatory network construction and key target screening, the corresponding targets of the key biologically active ingredients of Cortex Periplocae and the hepatocellular carcinoma-related targets are imported into Cytoscape 3.7.2 respectively, and the protein-protein interaction relationship network of the targets of the active ingredients of Cortex Periplocae and the hepatocellular carcinoma targets is constructed and interactively processed; through analysis, the key targets are determined, and 8 key targets are obtained: FOXO1, PTGS1, HSP90AA1, PTGS2, PRKCA, BCL2, NR3C1, and PGR.

10. The method for screening targets of periplocin and hepatocellular carcinoma according to claim 9, characterized in that: In the transcriptomic analysis and key target determination, transcriptomic research was conducted, total RNA of the control group and the medium concentration group of periplocin was extracted and detected, library construction was performed, transcriptomic RNA sequencing analysis (RNA-seq), differentially expressed mRNA was screened, and KEGG analysis was performed on it; aging-related targets were screened out using Geneage and GeneCard databases, and the intersection with sequencing data was obtained to construct a "target network". The node network centrality was evaluated according to the three topological characteristic indicators of BC, CC, and DC, and the key active ingredients in the network were screened, and FOXO1 was finally determined as a key target.

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