Application of periplocin in anti-lung adenocarcinoma medicine and method for screening target spot of periplocin

Abstract

The invention relates to application of periplocin in anti-lung adenocarcinoma drugs and a target screening method of periplocin. Periplocin can be used for preparing anti-cancer drugs for treating lung adenocarcinoma, and can inhibit proliferation, invasion and metastasis of lung adenocarcinoma cells and induce apoptosis of the lung adenocarcinoma cells. Meanwhile, the invention also provides a method for screening anti-lung adenocarcinoma related targets of periplocin by combining transcriptomics with network pharmacology, which comprises the following steps: firstly, searching effective active components and prediction action targets of periplocin through a TCMSP database, constructing network pharmacology to screen key components and targets, and then constructing and analyzing a regulatory network. Intersection genes are determined in combination with transcriptomics, and a new thought is provided for lung adenocarcinoma treatment and drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically to the application of periplocin in lung adenocarcinoma drugs and a method for screening targets thereof. Background Art

[0002] According to histopathological classification, lung cancer can be divided into two major categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Among them, lung adenocarcinoma (LUAD) is the main pathological type of non-small cell lung carcinoma (NSCLC). Although the treatment of LUAD has developed from traditional surgical treatment, chemotherapy and radiotherapy to precise molecular targeted therapy and immunotherapy in recent years, most patients will eventually develop drug resistance after treatment, and the 5-year survival rate is still less than 16.1%, and the efficacy is still unsatisfactory. Therefore, in-depth exploration of the molecular mechanism of lung adenocarcinoma and the search for new prevention and treatment targets and drugs are of great scientific significance and clinical practical value.

[0003] Traditional Chinese medicine is a valuable heritage of Chinese culture. Throughout China's long history, it has been proven to be an important tool for the prevention and treatment of diseases. Periplocin, also known as Northern Acanthopanax Cortex, Sheep Milk Vine, Sheep Peach Tip, Sheep Milk, and Periploca Cortex, belongs to the Asclepiadaceae family and has the effects of diuresis, detumescence, rheumatism, and strengthening bones and muscles. Periplocin, also known as Periplocin G and Periplocin, is an alpha-type cardiac glycoside with a steroid nucleus and an unsaturated five-membered lactone ring structure. It is one of the effective ingredients of Periploca Cortex for the treatment of chronic congestive heart failure and cardiac edema. As the main cardiotonic ingredient of Periploca Cortex for the treatment of heart failure, Periplocin has the characteristics of fast action speed and short duration. Previous studies have shown that Periplocin can significantly inhibit the proliferation of human hepatoma cells, arrest the cell cycle at the G2 / M phase, and induce cell apoptosis. Periplocin also has the activity of inhibiting the proliferation of esophageal squamous cell carcinoma cells and breast cancer cells. However, its anticancer effect has not been fully studied. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application of periplocin in anti-lung adenocarcinoma drugs and a method for screening targets thereof, so as to provide more and better options for the treatment of cancer, especially lung adenocarcinoma.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an application of periplocin in an anti-lung adenocarcinoma drug, the drug comprising an active component and a pharmaceutical excipient; the active component contains periplocin.

[0006] The present invention is further configured as follows: the medicine is used to treat lung adenocarcinoma.

[0007] The present invention is further configured as follows: the drug is used to inhibit the proliferation of lung adenocarcinoma cells, inhibit the invasion of lung adenocarcinoma cells, and induce the apoptosis of lung adenocarcinoma cells.

[0008] The present invention is further configured as follows: the lung adenocarcinoma cells are H2009, H1299 or A549 cells.

[0009] The present invention also provides a method for screening targets of periplocin and lung adenocarcinoma, comprising the following steps:

[0010] A. Database-based screening of active ingredients and target prediction;

[0011] B. Construction of active ingredient-target network and screening of key active ingredients;

[0012] C. Construction of regulatory network and screening of targets related to lung adenocarcinoma;

[0013] D. The intersection genes are determined.

[0014] In summary, the present invention has the following beneficial effects:

[0015] The present invention provides a new anticancer use of periplocin, which has a good anticancer effect on lung adenocarcinoma, especially lung adenocarcinoma.

[0016] At the same time, the combination of multiple technical means can accurately screen targets (such as SIRT5), providing a basis for understanding the mechanism of action of periplocin in lung adenocarcinoma. At the same time, the target can guide the development of lung adenocarcinoma drugs and improve the accuracy of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the result of network pharmacology combined with transcriptomics screening in Example 1 of the present invention.

[0018] Figure 2 This is the result that the periplocin in Example 2 of the present invention can inhibit the proliferation of lung adenocarcinoma cells.

[0019] Figure 3 These are the results of the effects of periplocin on the invasion and apoptosis of lung adenocarcinoma cells in Example 3 of the present invention.

[0020] Figure 4 This is the key target screening of periplocin against lung adenocarcinoma in Example 4 of the present invention.

[0021] Figure 5 In Example 5 of the present invention, periplocoside exerts its anti-lung adenocarcinoma effect by targeting SIRT5. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments.

[0023] MTT: The MTT method was used to detect the survival and mortality of human non-small cell lung cancer cells. NSCLE cells (9×103 cells / well) were seeded on 96-well plates and treated appropriately in different culture media. After 48 hours, MTT solution was added at a rate of 20 μL / well and cultured at a temperature of about 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 according to GraphPad ProPrism 8.0.

[0024] Colony formation assay: Human NSCLC cells (700-1000 cells / well) were seeded in six-well plates at 37°C in a 5% CO2 atmosphere overnight. Dimethyl sulfoxide (control), periwinkle glycosides (0.025, 0.1, 0.2 μM or 0.05, 0.2, 0.4 μM) were added to the cells for 48 hours. The culture medium was replaced with fresh culture medium every 3 days to maintain cell growth for 14 days. The colonies were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed 3 times with PBS, and stained with crystal violet for 10 minutes. Each experiment was performed three times, with a total of three independent experiments.

[0025] EdU experiment: EdU staining with BeyoClick to study cell proliferation TM EdU cell proliferation kit containing Alexa Fluor 555 (Beyotime, China, Cat. No. C0075S). First, wash the cells three times with PBS for five minutes each time. Add a certain amount of EDU to the fresh culture medium. The cells were cultured in a CO2 incubator at 37°C for 2 hours. After culture, wash the cells again with PBS three times for 5 minutes each time. The cells were fixed with 0.5% Triton X-100 at room temperature for 15 minutes, and then stained with Click reaction solution for 30 minutes. Wash again with PBS three times for 5 minutes each time, then stain with Hoechst33342 for 10 minutes, and wash again with PBS three times for 5 minutes each time. Observe the cells under an inverted fluorescence microscope.

[0026] TRANSWELL experiment: According to the manufacturer's instructions, a 24-well cell chamber with 8um pore size PC (polycarbonate) membrane (NEST) was used for the study to observe cell migration. Cells were added to the upper chamber containing serum-free medium at a density of 1×105. Medium containing 10% FBS was added to the lower chamber. After drug treatment for 48 hours, the cells were fixed with 4% paraformaldehyde and the unpulverized cells were removed from the upper chamber surface of the chamber. The cells on the lower surface of the membrane were stained with 1% crystal violet for 15 minutes at room temperature in the dark. The number of migrated cells was recorded and counted under an optical microscope. Images were taken under an inverted microscope.

[0027] Scratch assay: Cells (4 × 105 cells / well) were seeded on 6-well plates and allowed to adhere overnight. At 80–90% confluence, five “reference lines” were scratched on the bottom of the plate using a sterile 200 μL pipette tip. After washing three times with phosphate-buffered saline (PBS), cells were further incubated with inhibitors or controls (DMSO) in culture medium to examine cell migration in the absence of cell growth. Micrographs of cell migration across the reference lines were taken in different areas using an inverted phase-contrast microscope at 0, 24, and 48 hours after treatment. The migration rate was quantified by the distance of cell migration from the reference line to the center compared to the control group. All experiments were repeated 3 times.

[0028] Apoptosis assay: H2009, H1299, and A549 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), periplocin (0.025, 0.1, 0.2 μM, or 0.05, 0.2, 0.4 μM) for 48 h to evaluate the effects of these compounds on 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.

[0029] WB experiment: Cancer cells were seeded on 6-well plates at a density of 4×105 cells / well and then cultured overnight. Dimethyl sulfoxide (control), periplocin (0.025, 0.1, 0.2 μM or 0.05, 0.2, 0.4 μM) were added to the 6-well plates. The treated cells were washed with ice-cold PBS and lysed with 1% phenylmethylsulfonyl fluoride in RIPA lysis buffer (total protein extraction kit). After high-speed centrifugation to obtain the supernatant, the proteins were collected and quantified with an enhanced BCA protein assay kit. Subsequently, about 60 μg of protein was loaded on 8%, 10%, or 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and separated. Then, they were transferred to PVDF membranes. The membranes were washed three times with TBST and blocked with 5% fresh skim milk for 1.5 hours at room temperature. The blots were incubated with specific primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with relevant secondary antibodies for 60 minutes at room temperature. Immunoreactive bands were visualized using the Omni ECL femtosecond photochemiluminescence kit (EpiZyme, Shanghai, China). Visualization of the bands was analyzed using ImageJ computer software.

[0030] Example 1

[0031] In this example, all components of Cortex Periplocae were retrieved through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and 17 key bioactive components of Cortex Periplocae were obtained by screening based on the pharmacokinetic parameters of each component ( Figure 1 A). The TCMSP target prediction model was used to predict the targets of the above active ingredients, and duplicate targets were excluded, and a total of 38 predicted targets were obtained. The network visualization software Cytoscape3.7.2 was used to import the screened key active ingredients and their targets of Periploca radiata to construct the "active ingredient-target network" of Periploca radiata ( Figure 1B). The node network centrality was evaluated according to the three topological characteristic indicators of node betweenness centrality (BC), node closeness centrality (CC) and node degree of freedom (DC), and the top 10 key active ingredients in the "Cortex Periplocae Active Ingredients-Target" network were screened by sorting by DC. The targets corresponding to the key bioactive components of Cortex Periplocae and the targets related to lung adenocarcinoma were imported into the BisoGenet plug-in of Cytoscape 3.7.2, respectively. The protein-protein interaction relationship networks of the targets of the active components of Cortex Periplocae and the targets of lung adenocarcinoma were constructed and interactively processed, which were the direct or indirect target regulation networks of Cortex Periplocae in the treatment of lung adenocarcinoma. The CytoNCA plug-in was used to perform topological parameter analysis and screening on the above results. The important targets were evaluated using six parameters: BC, CC, DC eigenvector centrality (EC), network centrality (NC), and local average connectivity (LAC). The screening criteria were that DC was greater than 2 times the median, and BC, CC, EC, NC, and LAC were greater than 1 times the median ( Figure 1 C). Among the important targets, targets directly related to both composition and lung adenocarcinoma were selected as key targets. Through network topology analysis, a total of 9 key targets were found (PGR, PRKACA, JUN, SIRT5, PTGS2, GABRA1, CASP3, TGFB1, CASP9) ( Figure 1 D).

[0032] Example 2

[0033] This example shows that the MTT test experiment shows that periplocin has a dose-dependent and time-dependent cytotoxic and anti-proliferative effect on NSCLC cells ( Figure 2 B). The effect of glycosides on the growth of NSCLC cells was detected by EDU and clone formation assays. EDU cell proliferation assays showed that glycosides could inhibit the proliferation of NSCLC cells in a dose-dependent manner ( Figure 2 C); The results of the clone formation experiment showed that periplocin significantly inhibited the number of clones formed by H1299, A549 and H2009 cells ( Figure 2 D) These results suggest that periplocin can inhibit the proliferation of lung adenocarcinoma cells.

[0034] Example 3

[0035] This example further observes the effect of periplocin on the invasion and apoptosis of lung adenocarcinoma cells. In the TRANSWELL experiment, the addition of the drug greatly reduced the invasion and metastasis ability of H1299, A549 and H2009 cells ( Figure 3 A). The results of the scratch healing assay of H1299 and A549 cells further support the role of periplocin in metastasis ( Figure 3 B). 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 glycosides can promote apoptosis of H1299, A549 and H2009 cells ( Figure 3 C).

[0036] Example 4

[0037] In this example, total RNA was extracted and detected from the control group, the low-concentration group of periplocin, and the high-concentration group of periplocin, library construction was performed, and transcriptome RNA sequencing (RNA sequencing, RNA-seq) sequencing analysis was performed to screen out 348 differentially expressed mRNAs, of which 137 genes were up-regulated and 271 genes were down-regulated ( Figure 4 A), and GO functional enrichment analysis and KEGG signaling pathway enrichment analysis were performed on the differentially expressed genes, including P53 signaling pathway, cell aging-related pathway, herpes virus infection, MARK signaling pathway, etc. ( Figure 4 B). Using five databases, GeneCard, OMIM, PharmGkb, TTD, and DrugBank, lung cancer-related targets were screened and intersected with the predicted targets of the active ingredients of Periploca rotundus by TCMSP, resulting in 38 targets ( Figure 4 C). Transcriptome RNA sequencing analysis screened out 348 differentially expressed mRNAs, and Kaplan-Meier Plotter analysis showed that 10 genes (SIRT3, SIRT5, PLOD1, VSIG10L, PLOD1, DKK3, NXA10, TRADB, and VSIG10L) were prognosis-related genes. In network pharmacology, a total of 9 key targets (PGR, PRKACA, JUN, SIRT5, PTGS2, GABRA1, CASP3, TGFB1, and CASP9) were found. The differentially expressed genes screened by transcriptome sequencing were intersected with the genes screened in lung adenocarcinoma network pharmacology, and 1 intersection gene SIRT5 was obtained.

[0038] Example 5

[0039] In this example, a computer molecular docking simulation was performed through the Molecule Docking experiment. The results showed that the glycosides of Periploca quinata and the amino acids Asp236 and Glu263 of SIRT5 produced four hydrogen bonds with a distance of and binding energy: -5.759kcal / mol( Figure 5 A). This suggests that periwinkle glycosides may be a targeted inhibitor of SIRT5. This study further analyzed the expression differences of SIRT5 mRNA in lung adenocarcinoma tissues using the TCGA database. The results showed that SIRT5 mRNA expression was increased in lung adenocarcinoma tissues (P = 9e-07) ( Figure 5 B), and explored the role of SIRT5 expression in the prognosis of patients with lung adenocarcinoma. The Kaplan-Meier survival curve was drawn, and the results showed that the overall survival of patients with lung adenocarcinoma with high SIRT5 expression was shorter, and the difference was statistically significant (P=0.034) ( Figure 5 C). The above results suggest that SIRT5 may become a biomarker for poor prognosis of lung adenocarcinoma.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the design concept of the present invention should be included in the protection scope of the present invention.

Claims

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

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

3. The use of periplocin in anti-lung adenocarcinoma drugs according to claim 1, characterized in that: The medicine is used for inhibiting the proliferation of lung adenocarcinoma cells, inhibiting the invasion of lung adenocarcinoma cells, and inducing the apoptosis of lung adenocarcinoma cells.

4. The use of periplocin in anti-lung adenocarcinoma drugs according to claim 3, characterized in that: The lung adenocarcinoma cells are H2009, H1299 or A549 cells.

5. A method for screening targets of periplocin and lung adenocarcinoma, characterized in that: The following steps are involved: A. Screening of active drug ingredients and target prediction based on TCMSP database; B. Construction of the active ingredient-target network of periplocoside and screening of key active ingredients; C. Transcriptomics detection of targets of periplocoside; D. Construction of regulatory network and screening of targets related to lung adenocarcinoma; E. Based on the intersection genes of network pharmacology and transcriptomics, the targets of smilax glabra glycosides in lung adenocarcinoma were determined.

6. The method for screening targets of periplocin and lung adenocarcinoma according to claim 5, characterized in that: In the active ingredient screening and target prediction based on the TCMSP database, it includes: The active ingredients of the Chinese medicine Periploca odorata were searched using the TCMSP database; Screen out multiple key active pharmaceutical ingredients based on pharmacokinetic parameters; The target prediction model of the platform was used to predict the targets of these active ingredients, and a set of predicted targets was obtained after excluding duplicate targets.

7. The method for screening targets of periplocin and lung adenocarcinoma according to claim 6, characterized in that: In the construction of the active ingredient-target network and the screening of key active ingredients, it includes: The network visualization software Cytoscape3.7.2 was used to construct the network of the above-screened active ingredients and their targets; Including but not limited to evaluating the node network centrality based on the topological characteristic indicators of node betweenness centrality, node compactness and node freedom; Sort by node degrees of freedom to filter out the top key active ingredients.

8. The method for screening targets of periplocin and lung adenocarcinoma according to claim 7, characterized in that: In the construction of the regulatory network and the screening of targets related to lung adenocarcinoma, the following are included: The targets corresponding to the key bioactive ingredients and lung adenocarcinoma-related targets were imported into the BisoGenet plug-in of Cytoscape 3.7.2 to construct a protein-protein interaction relationship network and perform interactive processing to obtain a regulatory network; CytoNCA was used to analyze and screen the topological parameters of the network. Six parameters, including node betweenness centrality, node compactness, node degrees of freedom, eigenvector centrality, network centrality and local edge connectivity, were used to evaluate important targets. The screening criteria were that the node degrees of freedom were greater than 2 times the median, and the other five parameters were greater than 1 times the median. Among the important targets, those directly related to both components and lung adenocarcinoma were screened out.

9. The method for screening targets of periplocin and lung adenocarcinoma according to claim 8, characterized in that: In the determination of the intersection genes, it includes: The key targets obtained through network topology analysis were combined with transcriptome RNA sequencing analysis to screen out differentially expressed mRNAs; Prognosis-related genes were determined by Kaplan-Meier Plotter analysis; The differentially expressed genes screened by transcriptome sequencing were intersected with the genes screened by network pharmacology to obtain the intersection gene SIRT5.

Images