Traditional Chinese medicine composition for treating precancerous lesions of gastric cancer by inhibiting cell senescence

Abstract

The invention provides a traditional Chinese medicine composition for treating precancerous lesions of gastric cancer by inhibiting cell aging, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition provided by the invention can remove senescence cells, inhibit SASP-related inflammatory factors and reverse gastric cancer precancerous lesion pathological characteristics, reveals that the traditional Chinese medicine composition treats PLGC through a targeted cell senescence pathway for the first time, and fills the blank of traditional Chinese medicine mechanism research. According to the invention, network pharmacology, single cell sequencing and in-vivo and in-vitro models are integrated, so that scientificity and repeatability of the technical scheme are ensured, a safe and effective traditional Chinese medicine treatment scheme is provided for precancerous lesions of the gastric cancer, and the occurrence risk of the gastric cancer is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly to a traditional Chinese medicine composition for treating precancerous lesions of gastric cancer by inhibiting cell senescence. Background Art

[0002] Precancerous lesions of gastric cancer (PLGC) are the most common pathological states of gastric mucosa that progress to gastric adenocarcinoma. Globally, its incidence shows an upward trend, seriously threatening human health. Since there is currently no specific treatment method, many PLGC patients are at risk of the disease progressing to gastric adenocarcinoma, which not only brings a huge burden to the patients' physical and mental health, but also causes a heavy pressure on social medical resources.

[0003] In the process of researching and treating PLGC, although there are some traditional treatment ideas and methods, they all have limitations to varying degrees and cannot meet the needs of clinical treatment. For example, some treatment means have large side effects and poor patient tolerance; while other methods have insignificant curative effects and are difficult to effectively prevent the progression of PLGC to gastric adenocarcinoma.

[0004] Therefore, in-depth research on the pathogenesis of PLGC and finding safe and effective treatment methods and drugs have important clinical significance and social value for improving the prognosis of PLGC patients and reducing the incidence of gastric adenocarcinoma. Exploring new treatment targets and intervention strategies at the molecular mechanism level is expected to bring new breakthroughs in the treatment of PLGC and new hopes to the majority of patients. Summary of the Invention

[0005] The purpose of the present invention is to provide a traditional Chinese medicine composition for treating precancerous lesions of gastric cancer by inhibiting cell senescence, which solves the problem of the lack of specific treatment methods for precancerous lesions of gastric cancer at present.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a traditional Chinese medicine composition, which contains the following raw materials by weight:

[0008] 13 - 17 parts of Artemisia capillaris Thunb., 10 - 14 parts of Scutellaria baicalensis Georgi, 13 - 17 parts of Hedyotis diffusa Willd., 13 - 17 parts of Isatis tinctoria L., 13 - 17 parts of Lobelia chinensis Lour., 7 - 11 parts of Pogostemon cablin (Blanco) Benth., 13 - 17 parts of Scutellaria barbata D. Don, 8 - 12 parts of Sophora flavescens Ait., 10 - 14 parts of Coptis chinensis Franch., 13 - 17 parts of Gynostemma pentaphyllum (Thunb.) Makino, 7 - 11 parts of Eupatorium fortunei Turcz.

[0009] Preferably, the dosage form of the traditional Chinese medicine composition is selected from tablets, pills, capsules, granules, powders, oral liquids, syrups, ointments or aerosols.

[0010] Preferably, the traditional Chinese medicine prescription further includes pharmaceutically acceptable excipients.

[0011] The present invention also provides the use of the above traditional Chinese medicine composition in the preparation of a medicament for treating precancerous lesions of gastric cancer.

[0012] The present invention also provides a preparation method of the above traditional Chinese medicine composition, comprising the following steps:

[0013] Mix all the raw materials with water, decoct and concentrate to obtain the traditional Chinese medicine composition.

[0014] Preferably, the weight ratio of the raw materials to water is 1:5 to 10.

[0015] Preferably, the concentration multiple of the concentration is 30 to 40 times.

[0016] Preferably, the decoction includes boiling for 5 to 15 minutes and then slow-fire decoction.

[0017] The present invention also provides the use of the above traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above preparation method in the preparation of an aging cell scavenging reagent.

[0018] The present invention also provides the use of the above traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above preparation method in the preparation of a reagent for regulating the senescence-associated secretory phenotype.

[0019] Advantages of the present invention:

[0020] The traditional Chinese medicine composition provided by the present invention can scavenge senescent cells, inhibit SASP-related inflammatory factors, and reverse the pathological characteristics of precancerous lesions of gastric cancer. The present invention first reveals that the traditional Chinese medicine composition treats PLGC by targeting the cell senescence pathway, filling the gap in the research on the mechanism of traditional Chinese medicine. The present invention integrates network pharmacology, single-cell sequencing, and in vitro and in vivo models to ensure the scientificity and reproducibility of the technical solution, provides a safe and effective traditional Chinese medicine treatment plan for precancerous lesions of gastric cancer, and reduces the risk of gastric cancer occurrence. Description of the drawings

[0021] Figure 1 is the experimental flow chart of the embodiment of the present invention;

[0022] Figure 2 are: (A) compound-target network diagram, where the V shape represents the compound and the rectangle represents the target; (B) PCA diagrams of three data sets; (C) volcano diagrams of three data sets;

[0023] Figure 3as follows: (A) Venn diagram, where GEO represents differentially expressed genes from external datasets and GeneCards represents disease targets from the GeneCards database; (B) protein-protein interaction (PPI) network of central targets, with the size of the circles proportional to the degree of connection; (C) bar chart showing the results of Gene Ontology (GO) analysis; (D) bubble chart illustrating the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis; (E) network diagram of pathways, targets, and components related to cellular senescence when HZJD treats PLGC;

[0024] Figure 4 as follows: (A) heatmap showing the results of molecular docking; (B) schematic diagram of the molecular docking results;

[0025] Figure 5 as follows: (A) UMAP plot of gastric tissue; (B) cell proportion plot, with the same legends for A and B; (C) violin and box plots depicting HZJD-related senescence genes; (D) UMAP plot of epithelial cells; (E) cell proportion plot, with a common legend for both; (F) GSEA analysis results comparing PLGC cells with gastric mucosal cells;

[0026] Figure 6 as follows: (A) schematic diagram of the establishment method of the PLGC rat model; (B) body weight curves of rats in different groups; (C) HE, AB-PAS, and SA-β-gal staining results, as well as immunofluorescence staining of MUC2 and P16; (D) HE histopathological score; (E) AB-PAS staining score; (F) SA-β-gal staining area; (G) immunofluorescence staining density of MUC2 and P16; (H) RT-qPCR results of p16, p21, p53, IL-6, NF-κB, and TNF; (I) Western blot detection results; (J) protein expression results of p16, p21, and p53; # indicates significant difference compared with the control group (# indicates p < 0.05, ## indicates p < 0.01, indicates p < 0.001, ns indicates p > 0.05); * indicates significant difference compared with the model group (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates p > 0.05);

[0027] Figure 7were: (A) Results of SA-β-gal staining and P16 immunofluorescence staining; (B) Quantification of SA-β-gal staining area; (C) RT-qPCR results of p16, p21, p53, IL-6, NF-κB and TNF; (D) Quantitative analysis of P16 immunofluorescence staining intensity; (E and F) Western blot results of p16, p21 and p53 protein expression; # indicates significant difference compared with the control group GES-1 (p<0.05 for #, p<0.01 for ##, p<0.001 for ); * indicates significant difference compared with the model group GES-1+D-gal or MC (p<0.05 for *, p<0.01 for **, p<0.001 for ***);

[0028] Figure 8 was the mass spectrometry analysis result diagram, with the anode on the top and the cathode on the bottom in the figure;

[0029] Figure 9 were: (A) Violin plots of biomarkers of each cell subset; (B) Dot plots of markers of each cell subset; (C) GSEA analysis results of NAG and PLGC epithelial cells;

[0030] Figure 10 were: (A) Violin plots of biomarkers of each cell subset; (B) Dot plots of markers of each cell subset;

[0031] Figure 11 were: (A) CCK-8 results after 24-hour MNNG intervention; (B) Morphology of GES-1 and MC cells under bright-field microscope; (C) CCK-8 results of MC cells after 24-hour HZJD treatment; (D) CCK-8 results of MC cells after 48-hour HZJD treatment; (E and F) P16 results of GES-1 cells treated with different concentrations of D-gal; (G and H) Wound healing assay results; (I) CCK-8 cell viability detection after 24 hours; # indicates significant difference compared with the control group GES-1 (p<0.05 for #, p<0.01 for ##, p<0.001 for ); * indicates significant difference compared with the model group GES-1+D-gal or MC (**p<0.05, **p<0.01, ***p<0.001). Specific implementation manners

[0032] The present invention provides a traditional Chinese medicine composition, which contains the following raw medicinal materials by weight parts:

[0033] 13 - 17 parts of Artemisia capillaris Thunb., 10 - 14 parts of Scutellaria baicalensis Georgi, 13 - 17 parts of Hedyotis diffusa Willd., 13 - 17 parts of Isatis tinctoria L., 13 - 17 parts of Lobelia chinensis Lour., 7 - 11 parts of Pogostemon cablin (Blanco) Benth., 13 - 17 parts of Scutellaria barbata D. Don, 8 - 12 parts of Sophora flavescens Aiton, 10 - 14 parts of Coptis chinensis Franch., 13 - 17 parts of Gynostemma pentaphyllum (Thunb.) Makino, 7 - 11 parts of Eupatorium fortunei Turcz.

[0034] Preferably, the dosage form of the traditional Chinese medicine composition is selected from tablets, pills, capsules, granules, powders, oral liquids, syrups, ointments or aerosols. Preferably, the traditional Chinese medicine formula further comprises pharmaceutically acceptable excipients.

[0035] The present invention also provides the use of the above traditional Chinese medicine composition in the preparation of a medicament for treating precancerous lesions of gastric cancer.

[0036] The present invention also provides a preparation method of the above traditional Chinese medicine composition, comprising the following steps: mixing all the raw materials with water, decocting and concentrating to obtain the traditional Chinese medicine composition.

[0037] Preferably, the weight ratio of the raw materials to water is 1:5 - 10. Preferably, the concentration multiple of the concentration is 30 - 40 times. Preferably, the decoction includes boiling for 5 - 15 min and then slow - fire decocting.

[0038] The present invention also provides the use of the above traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above preparation method in the preparation of a senescent cell scavenging reagent, and the senescent cell scavenging reagent can reduce the number of senescent cells in tissues or organisms.

[0039] The present invention also provides the use of the above traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above preparation method in the preparation of a reagent for regulating the senescence - associated secretory phenotype (SASP). The SASP includes IL - 6, IL - 8, TNF, NFκB, CCL2, CXCL1, etc. SASP has certain pathogenicity, can drive secondary senescence, destroy tissue homeostasis, and cause tissues to lose the ability of repair and regeneration. In the present invention, in MNNG - induced PLGC rats and MC cells, the expression levels of SASP components such as IL - 6, NF - κB and TNF increase, while the traditional Chinese medicine composition provided by the present invention can reverse this phenomenon.

[0040] The following combines examples to elaborate in detail on the technical solutions provided by the present invention, but they cannot be construed as limiting the protection scope of the present invention.

[0041] Examples

[0042] Traditional Chinese medicine granules: 15 g of Artemisia capillaris, 12 g of Scutellaria baicalensis, 15 g of Hedyotis diffusa, 15 g of Isatis tinctoria, 15 g of Lobelia chinensis, 9 g of Pogostemon cablin, 15 g of Scutellaria barbata, 10 g of Sophora flavescens, 12 g of Coptis chinensis, 15 g of Gynostemma pentaphyllum, and 9 g of Eupatorium fortunei, all from China Shineway Pharmaceutical Group Co., Ltd. (Shijiazhuang, China).

[0043] Mix all the granules, add 1000 ml of pure water, boil for 10 minutes, and finally concentrate by decocting over a slow fire to 30 ml to obtain the product.

[0044] Ultra-high performance liquid chromatography - quadrupole - time-of-flight - tandem mass spectrometry (UPLC-Q-TOF-MS / MS) analysis

[0045] Chromatographic separation was performed using an UltiMate 3000 UPLC system (Thermo Fisher Scientific, Bremen, Germany). Reverse-phase separation was carried out on an ACQUITY UPLC T3 column (100 mm * 2.1 mm, 1.8 μm, Waters, Milford, MA, USA). The column oven was maintained at 40 °C, and then solvent A (5 mM ammonium acetate and 5 mM acetic acid) and solvent B (acetonitrile) were added. The low flow rate was 0.3 ml / min. The mobile phase was solvent A. The gradient elution conditions were set as follows: 0 - 2.8 min, 2% B; 2.8 - 5.6 min, 70% B; 5.6 - 6.4 min, 90% B; 6.4 - 8.0 min, 100% B; 8.0 - 8.1 min, 100% B; 8.1 - 10 min, 2% B. The high-resolution mass spectrometer used was a TripleTOF 6600 (SCIEX, Framingham, MA, USA) time-of-flight mass spectrometer. Each sample was collected once in the positive ion mode and once in the negative ion mode. The shield gas pressure of the ion source was 30 PSI (pounds per square inch), and the pressures of both gas 1 (auxiliary gas) and gas 2 (sheath gas) were set to 60 PSI. The source temperature was 500 °C. The ion source pressure was set to 60 PSI in the positive ion mode. The voltage was +5000 V in the positive ion mode and -4500 V in the negative ion mode. The data collection mode was IDA (information-dependent acquisition) mode. In one acquisition cycle, the primary acquisition range was 60 - 1200 daltons, and the primary acquisition time was 150 ms. Then, the top 12 signal ions with a signal accumulation intensity exceeding 100 were selected from the primary spectrum for secondary fragmentation scanning. The dynamic exclusion was set to 4 s. During the acquisition process, the instrument accuracy calibration was performed every 20 sample intervals. At the same time, a quality control (QC) product scan was performed every 10 sample intervals. The mass gap between QCs was used to correct the systematic error of the entire batch of experiments. Finally, the data was collected and analyzed using XCMS software. The mass spectrometry analysis results are shown in Figure 8 .

[0046] Component and target identification of the traditional Chinese medicine composition provided by the present invention

[0047] The components with high relative abundances obtained by UPLC-MS were input into the PubChem database 3.10.1 software (https: / / pubchem.ncbi.nlm.nih.gov / ) to obtain the corresponding smiles numbers and 2D structures. Then, ADME analysis was performed through SwissADME (http: / / www.swissadme.ch / ) to screen for active compounds. The screening criteria were "high" gastrointestinal absorption and "yes" for two or more of Lipinski, Ghose, Veber, Egan, and Muegge. Finally, target prediction was performed on the screened active compounds using SwissTargetPrediction (http: / / swisstargetprediction.ch / ), and the target screening criterion was Probability > 0.12. At the same time, Cytoscape (Cytoscape Consortium, California, USA) was used to visualize the screened active compounds and related targets.

[0048] Collection of the PLGC dataset

[0049] Three PLGC datasets were retrieved from the GEO database: GSE2741123, GSE7852324, GSE13082325, and GSE13452026. GSE27411 contains microarray data of 12 CNAG tissue samples and 6 CAG tissue samples. GSE78523 is also a microarray dataset, including 15 healthy individual samples and 30 IM tissue samples. GSE130823 is another microarray dataset, including 47 CNAG tissue samples, 31 intraepithelial neoplasia tissue samples, and 16 GC tissue samples. GSE134520 is a single-cell RNA-seq dataset, including 3 CNAG tissue samples, 3 CAG tissue samples, 6 IM tissue samples, and 1 GC tissue sample. In subsequent analyses, CNAG or healthy individuals were regarded as the control group, while CAG, IM, and intraepithelial neoplasia were classified into the PLGC group.

[0050] Microarray and single-cell RNA-seq data analysis

[0051] For microarray data analysis, the present invention uses the limma package in R to evaluate the differences in linear model fitting. Differentially expressed genes are selected based on an adjusted P-value less than 0.05 and an absolute log fold change (|logFC|) greater than 1. For single-cell RNA-seq data, the present invention uses the Seurat R package to integrate data from three CNAG samples and nine PLGC samples. Cells with 400 to 7000 genes expressed and a mitochondrial or ribosomal gene content less than 20% are filtered, and a total of 20,632 cells are obtained for further analysis. The present invention uses the NormalizeData, FindVariableFeatures, and ScaleData functions in Seurat to obtain a low-dimensional representation in the UMAP space. The FindAllMarkers and FindMarkers functions are used to identify the markers of each cell subset and the differentially expressed genes between the designated subsets, preparing for subsequent functional analysis.

[0052] Collection of PLGC-related targets and identification of intersection targets

[0053] The GeneCard database (https: / / www.genecards.org / ) is used to collect disease targets related to "precancerous lesions of gastric cancer". The screening criterion is a correlation score > 10. Subsequently, the VennDiagram package in R is used to explore the relationship between the targets related to the traditional Chinese medicine composition provided by the present invention and the PLGC-related targets, so as to obtain intersection targets and draw a Venn diagram.

[0054] Construction and analysis of protein-protein interaction networks

[0055] The protein-protein interaction (PPI) network system analyzes the interactions of a large number of proteins in biological systems. The String database (https: / / cn.string-db.org / ) is used for PPI analysis, and the screening criteria are an interaction score > 0.4 and disconnected nodes. Subsequently, a file in TSV format is generated and imported into Cytoscape software for network analysis.

[0056] Functional annotation and gene set enrichment analysis

[0057] The present invention uses the clusterProfiler package in R for enrichment analysis, including biological processes (BP), cellular components (CC), and molecular functions (MF) of gene ontology (GO) terms, as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Gene set enrichment analysis (GSEA) is performed on KEGG pathways and Hallmark gene sets, and the results are corrected for multiple testing using the false discovery rate (FDR) method.

[0058] Molecular docking analysis

[0059] Molecular docking is a research method that uses computer simulation to study the interaction between ligands and proteins. The 3D structures of hub genes and core compounds were downloaded from the Collaborative Structural Biology Protein Data Bank (https: / / www.rcsb.org / ) and the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), respectively. CB-Dock2 is a blind docking server that significantly improves the docking method by combining a template-based docking engine, enhances the accuracy of binding site recognition and binding pose prediction, and has been widely used in drug and biological research.

[0060] Animal model

[0061] The animal care and experimental procedures in this invention have been approved by the Institutional Animal Care and Use Committee of Hebei Provincial Hospital of Traditional Chinese Medicine (IACUC-HPHCM-2024087) and comply with international regulations for laboratory animal management. Male Sprague-Dawley (SD) rats (150 - 180 g) used in this experiment were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License number: SCXK(Beijing)2019 - 0008). All rats were housed in an animal laboratory at 20°C - 25°C, 40% - 70% humidity, with a 12-hour light / 12-hour dark cycle and provided with standard rat feed.

[0062] Establishment of the PLGC animal model

[0063] After one week of adaptive feeding, the rats were randomly divided into a control group and a model group. During the modeling period, the rats in the NC group drank clean water and had a normal diet freely. The model rats were modeled using a combined modeling method of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), which is a mature modeling method established by the research team of this invention. The model rats were given 200 μg / ml of MNNG (MedChemExpress, HY-128612, USA) every day and subjected to a starvation-feeding switch every other day.

[0064] Hematoxylin-eosin staining and Alcian blue-periodic acid Schiff staining

[0065] In the present invention, fresh gastric tissues were obtained from rats and immersed in 4% formaldehyde solution for 24 hours to ensure sufficient fixation. Then, the tissues were processed through a graded ethanol dehydration series and clarified with xylene to enhance transparency. Finally, they were embedded in paraffin to maintain tissue integrity. The paraffin-embedded blocks were cut into 5-micron-thick tissue sections. For hematoxylin and eosin (H&E) staining, the sections went through a meticulous protocol, including hematoxylin staining, differentiation with hydrochloric acid alcohol solution, staining with graded series of eosin, ethanol dehydration, xylene clarification, and finally mounting with neutral Canada balsam to protect and stabilize the stained sections. For alcian blue-periodic acid Schiff (AB-PAS) staining, the present invention followed the manufacturer's instructions provided by the kit (Servicebio, G1049, China). The staining procedure started with the application of AB-PAS staining solution C, then acidification with AB-PAS staining solution B, and subsequent staining with AB-PAS staining solution A. Then, the sections were dehydrated with a graded ethanol series, clarified with xylene, and finally mounted with neutral Canada balsam to maintain the integrity and visual quality of the stained preparation.

[0066] Senescence-associated β-galactosidase staining

[0067] The present invention used a senescence-associated β-galactosidase (SA-β-Gal) staining kit (Solarbio, G1580, China) to evaluate the distribution of senescent cells. According to the manufacturer's protocol, the present invention fixed fresh frozen sections or cells with β-Gal fixative, rinsed them with PBS, and then prepared a working solution with X-Gal and β-Gal staining solutions B, C, and D. After incubating overnight at 37 °C, the present invention observed the results under an optical microscope.

[0068] Cell culture

[0069] The GES-1 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Procell, China) and cultured in RPMI-1640 (Gibco, C11875500BT, USA) medium supplemented with 10% fetal bovine serum (Gibco, A5669701, USA) and 1% penicillin / streptomycin (Gibco, 15140-122, USA) in a humidified environment at 37°C with 5% CO2. When constructing the MC cell model, the present invention followed the protocol established by the research team of the present invention previously. The GES-1 cells were incubated in complete medium containing MNNG (MedChemExpress, HY-128612, USA) for 24 hours. Subsequently, the medium containing MNNG was removed, and the cells were cultured in RPMI-1640 medium without MNNG. During this period, a large number of cells detached and died, and when the cells reached 80-90% confluence, they were passaged, and the surviving cells showing a disordered growth pattern were passaged. The obtained cell model was called MC cells, and the third generation of these cells was used for subsequent experimental procedures. For the induction of the cell senescence model, the GES-1 cells were incubated in complete medium containing D-galactose (D-gal; MedChemExpress, HY-N0210, USA) for 48 hours. After incubation, the medium containing D-gal was removed, and the cells were further cultured in RPMI-1640 medium without MNNG to study cell senescence.

[0070] Preparation of serum containing drugs

[0071] Male SD rats (150-180 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (License number: SCXK-001) and housed in an animal laboratory under controlled conditions: temperature 20°C - 25°C, humidity 40% - 70%, 12-hour light / dark cycle. They were fed with standard rat feed. The preparation of serum containing drugs followed the method used in the previous research of the present invention, and they were gavaged twice a day for seven consecutive days. After anesthesia with sodium pentobarbital, blood was collected from bilateral femoral arteries. The blood was allowed to clot at room temperature for 2 hours, and then the serum was separated by centrifugation. The complement was inactivated by incubating in a water bath at 56°C. Finally, the serum containing the traditional Chinese medicine composition provided by the present invention was filtered through a 0.22 μm sterile filter, sterilized, and stored at -20°C for later use.

[0072] Cell viability assay

[0073] Cell viability was determined using a CCK-8 kit (MedChemExpress, HY-K0301, USA). 5×10^3 cells were seeded into 96-well plates and allowed to adhere. After adhesion, the cells were treated with different concentrations of MNNG and the medicated serum containing the traditional Chinese medicine composition provided by the present invention for a certain period of time. The supernatant was aspirated, and the CCK-8 reagent was diluted with the medium at a ratio of 1:10 to prepare a working solution. Then, the cells were incubated in a cell culture incubator for 2 hours in a light-protected environment. After incubation, the optical density (OD) of the cells at 450 nm was measured using a microplate reader. Subsequently, the cell viability was determined by comparing the obtained OD values.

[0074] Cell scratch assay

[0075] Cells were seeded into six-well plates at a concentration of 5×10^5 cells / well and allowed to adhere until 100% confluence was reached in the bottom area of the wells. A linear wound was created on the confluent cell monolayer using a sterile micropipette tip. After creating the wound, the wells were rinsed three times with phosphate-buffered saline (PBS) to remove any detached cells. Then, the medium was refreshed with serum-free medium. The migration of cells into the wound area was monitored and photographed by microscopy at three different time intervals of 0, 24, and 48 hours.

[0076] Transcription level detection

[0077] The transcription level of genes was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from gastric tissues or cultured cells using a total RNA extraction kit (Solarbio, R1200, China). Reverse transcription of mRNA into cDNA was performed using a Super plus qPCR RT kit combined with a gDNA removal kit (Mei5bio, MF166-plus, China), which also facilitated the removal of genomic DNA (gDNA). Then, quantitative PCR was performed using a HiPer SYBR Premix EsTaq kit (Mei5bio, MF787, China). The relative quantification of mRNA expression levels between different groups was calculated using the 2^-ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for the normalization of target gene expression.

[0078] Protein electrophoresis

[0079] Protein expression levels were evaluated by Western blot analysis. Total cellular proteins were extracted using RIPA buffer and protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Scientific, 23225, USA). Proteins were resolved by SDS-PAGE electrophoresis under constant voltage conditions and transferred to a PVDF membrane (Invitrogen, T2234, USA). Then, the membrane was blocked with non-fat milk powder (Solarbio, D8340, China). Primary antibodies were applied and incubated overnight at 4 °C. Subsequently, incubation with the corresponding horseradish peroxidase-conjugated secondary antibody was performed. Immunoreactive bands were quantified by densitometry using ImageJ software.

[0080] Immunofluorescence staining

[0081] Tissue specimens were cut into paraffin-embedded sections or cells were prepared as smear slides, and antigen retrieval was performed using citrate buffer (Servicebio, G1202, China). Subsequently, the samples were incubated with primary antibodies at 4 °C. Then, the sections were rinsed with phosphate-buffered saline (PBS) (Solarbio, P1010, China) and incubated with a horseradish peroxidase (HRP)-conjugated fluorescent secondary antibody in an opaque plastic container at room temperature to avoid light exposure. Finally, immunofluorescence was observed using a fluorescence microscope and images were acquired.

[0082] Statistical analysis

[0083] All experimental data in this invention were derived from at least three independent replicates and were expressed as mean ± standard deviation (SD). To compare variances between groups, one-way analysis of variance (ANOVA) using GraphPad Prism software was performed in this invention, supplemented with Tukey's multiple comparison test. The statistical significance of all pairwise comparisons was determined at a threshold of p < 0.05.

[0084] Results:

[0085] Identification of the drug targets related to the traditional Chinese medicine composition provided by this invention and the PLGC-related targets

[0086] This invention first used UPLC-Q-TOF-MS / MS for compound identification. After selecting high-abundance compounds, a total of 303 compounds were identified, including key compounds such as berberine, luteolin, and apigenin ( Figure 1) Subsequently, the present invention used the SwissADME platform to screen these compounds and obtained 212 active compounds. After initially identifying the compounds using UPLC-Q-TOF-MS / MS, the present invention used the SwissTargetPrediction platform to predict the targets of the active compounds. After a rigorous screening process, the present invention identified a total of 397 drug-related targets. Based on these targets, the present invention constructed a compound-target network containing 536 nodes and 1928 edges ( Figure 2 A). Topological analysis showed that the top five compounds ranked by degree value were luteolin, apigenin, isorhamnetin, 18-β-glycyrrhetinic acid (enoximone), and apigenin, indicating that these compounds may play a key role in disease treatment.

[0087] The present invention initially collected targets using the GeneCards database and obtained a total of 1857 targets related to precancerous lesions of gastric cancer (PLGC). To obtain a more comprehensive set of potential targets for PLGC, the present invention performed differential analysis on GSE27411, GSE78523, and GSE130823 using public databases ( Figure 2 B), and obtained 136, 308, and 1146 differentially expressed genes respectively ( Figure 2 C) for supplementation. After removing duplicates, the present invention obtained a total of 1490 unique differentially expressed genes.

[0088] Identification and functional analysis of hub targets

[0089] First, by mapping the genes in GeneCards, the differentially expressed genes obtained from the GEO public database, and the targets related to the traditional Chinese medicine composition provided by the present invention, the present invention identified a total of 181 hub targets ( Figure 3 A). Subsequently, the present invention used the String platform to construct a PPI network to clarify the connections between the 181 hub targets ( Figure 3 B). In the network, AKT1, IL6, TNF, ESR1, and EGFR were identified as the central nodes. Notably, IL6 and TNF are closely related to inflammation and immunity, indicating that the therapeutic effect of the traditional Chinese medicine composition provided by the present invention on PLGC may be closely related to inflammation regulation.

[0090] To gain an in-depth understanding of the mechanism of the therapeutic effect of the traditional Chinese medicine composition provided by the present invention on PLGC, the present invention performed GO and KEGG pathway enrichment analysis on the hub targets. GO analysis was mainly enriched in serine / threonine protein kinases, transferase complexes, and responses to external stimuli and inflammation, indicating that the treatment of PLGC with the traditional Chinese medicine composition provided by the present invention may be related to energy metabolism, cell cycle, inflammation, and DNA repair ( Figure 3C). The KEGG results were mainly enriched in signal transduction, cellular processes, the endocrine system, the immune system, and metabolic system pathways. Figure 3 D). It is worth noting that signal transduction is mainly related to the PI3K / AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, Foxo signaling pathway, and TNF signaling pathway. Cellular processes are mainly related to cellular senescence, focal adhesion, cell cycle, efferocytosis, and adherens junction. The endocrine system is mainly involved in progesterone-mediated oocyte maturation, ovarian steroidogenesis, prolactin signaling pathway, relaxin signaling pathway, and estrogen signaling pathway. The immune system is mainly involved in the IL-17 signaling pathway, C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, chemokine signaling pathway, and T cell receptor signaling pathway. The metabolic system is mainly involved in steroid hormone biosynthesis, nitrogen metabolism, arachidonic acid metabolism, cytochrome P450 metabolism of xenobiotics, and tryptophan metabolism. Notably, cellular senescence is the pathway with the highest count value apart from signal transduction-related pathways, and other pathways such as the cell cycle, PI3K / Akt signaling pathway, RAS signaling pathway, TNF signaling pathway, and IL-17 signaling pathway are all closely related to cellular senescence. This indicates that cellular senescence may be the key pathway for the traditional Chinese medicine composition provided by the present invention to treat PLGC. Studying the role of cellular senescence in the therapeutic effect of the traditional Chinese medicine composition provided by the present invention is of great significance for understanding the potential treatment strategies for PLGC.

[0091] Subsequently, the present invention established a network connecting the hub targets related to cellular senescence with the active compounds in the traditional Chinese medicine composition provided by the present invention to identify the bioactive components related to cellular senescence in the traditional Chinese medicine composition provided by the present invention. Figure 3 E). The bioactive components with the highest degree values are luteolin, isorhamnetin, apigenin, and 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-3,6-dimethoxy-4H-chromen-4-one (Bonanzin). Recent studies have shown that luteolin and isorhamnetin may be involved in cellular senescence. Luteolin has been shown to inhibit TNF-α-induced inflammation and cellular senescence in nucleus pulposus cells by regulating the Sirt6 / NF-κB pathway. Studies have also shown that luteolin can effectively prevent oxidative stress-induced cellular senescence through the p53 and SIRT1 pathways. In addition, isorhamnetin has been reported to prevent senescence, apoptosis, and oxidative stress damage of 6-OHDA-induced SH-SY5Y cells by targeting FOSL1 and activating the AKT / mTOR signaling pathway. To further confirm the connection between the traditional Chinese medicine composition provided by the present invention and cellular senescence, the present invention selected the top-ranked active components and the hub targets related to cellular senescence for molecular docking analysis. The docking results showed good binding affinity between these active components and the cellular senescence-related targets, with an average docking score of -0.733. Figure 4A and B). The pair with the highest absolute docking score is isorhamnetin and CDK2. This indicates that the therapeutic effect of the traditional Chinese medicine composition provided by the present invention on PLGC may be related to the regulation of cellular senescence.

[0092] Single-cell data analysis of the expression of cellular senescence-related hub targets

[0093] To further investigate the role of cellular senescence in PLGC and the effect of the traditional Chinese medicine composition provided by the present invention on it, the present invention analyzed the single-cell RNA-seq dataset GSE134520. After quality control, the cells were divided into eight subpopulations, and based on the expression of biomarkers EPCAM, CD79A, CD3D, LUM, ENG, CSF1R, TPSAB1, and ACTA2, these subpopulations were annotated as epithelial cells, B cells, T cells, fibroblasts, endothelial cells, macrophages, mast cells, and smooth muscle cells ( Figure 5 A and Figure 9 A). Meanwhile, the present invention used the FindAllMarkers function to identify the markers of each cell subpopulation ( Figure 9 B). Analysis of the proportions of various cell subpopulations showed that the proportion of immune cells increased and the proportion of epithelial cells decreased relatively in the PLGC stage compared with the NAG stage ( Figure 5 B), indicating an upregulation of the immune expression level in the PLGC stage. Subsequently, the present invention used the AddModuleScore function to score the cellular senescence-related hub targets in the treatment of PLGC with the traditional Chinese medicine composition provided by the present invention. The findings of the present invention showed that there were significant differences in the levels of cellular senescence-related hub targets between the NAG group and the PLGC group in epithelial cells, especially in the epithelial cells of the PLGC group where these targets were significantly upregulated ( Figure 5 C). This indicates that the cellular senescence-related hub targets show differential expression when entering the PLGC stage. Subsequently, the present invention used GSEA to examine the expression of KEGG pathways in epithelial cells of the NAG group and the PLGC group. Next, to further investigate the pathway differences between the NAG group and the PLGC group, the present invention used GSEA to analyze the expression of KEGG pathways in the two cohorts. The results showed that compared with the NAG stage, the pathways involved in the digestion and absorption of proteins, lipids, vitamins, carbohydrates, and minerals, as well as the bile secretion pathway and the complement and coagulation cascade pathway, were upregulated in the PLGC stage ( Figure 9 C).

[0094] To further clarify the changes in epithelial cells, the present invention isolated epithelial cells and further refined the analysis of the present invention by dividing them into six subpopulations. The markers used for this classification were MUC1, MUC6, OLFM4, SPINK4, CHGB, and FABP2 ( Figure 5 D andFigure 10 A). The present invention uses the FindAllMarkers function to identify the markers of these subpopulations ( Figure 10 B). By comparing the proportions of cell types, the present invention observes that compared with the NAG stage, the proportion of gastric mucosal cells decreases at the PLGC stage, while the proportions of PLGC cells, enteroendocrine cells, intestinal-like cells, SPEM cells, and goblet cells increase ( Figure 5 E). In addition, the present invention uses GSEA to compare the signature pathways between PLGC cells and normal gastric mucosal cells. The results show that compared with normal gastric mucosal cells, the oxidative phosphorylation, Myc target V1, and Myc target V2 pathways are upregulated in PLGC cells, while the E2f target, myogenesis, epithelial-mesenchymal transition, Tgf Beta signaling, Notch signaling, mitotic spindle, G2m checkpoint, and Tnfa are downregulated through the Nfkb signaling pathway ( Figure 5 F). Notably, previous studies have shown that the downregulation of the E2F target pathway may promote cellular senescence by affecting cell cycle progression and DNA damage response, while the downregulation of the G2M checkpoint pathway may be related to cell cycle regulation and DNA damage repair, thus affecting cellular senescence. These findings suggest that at the PLGC stage, especially in PLGC cells, it may be related to the expression of cellular senescence.

[0095] The traditional Chinese medicine composition provided by the present invention improves the pathological conditions of MNNG-induced PLGC rats and inhibits cellular senescence

[0096] To gain an in-depth understanding of whether the traditional Chinese medicine composition provided by the present invention for treating PLGC is related to cellular senescence, the present invention uses an MNNG-induced PLGC rat model to evaluate the therapeutic effect of the traditional Chinese medicine composition provided by the present invention and monitor the expression of cellular senescence markers ( Figure 6 A). The results show that after MNNG intervention, the body weight of the rats decreases, while the treatment with the traditional Chinese medicine composition provided by the present invention significantly reverses this effect, while FA treatment has no significant effect ( Figure 6 B). HE staining shows that the gastric mucosa of the control group rats appears normal, the epithelial cells are intact, and the glands are arranged in an orderly and compact manner without edema, congestion, or inflammatory infiltration. In contrast, the model group induced by MNNG shows disordered gland arrangement, gland atrophy, a small number of goblet cells, and inflammatory infiltration. These conditions are improved after intervention with the traditional Chinese medicine composition provided by the present invention or FA, and the effect of the group treated with the traditional Chinese medicine composition provided by the present invention is more obvious ( Figure 6 C and D). Similarly, the AB-PAS staining results show that compared with the control group, the blue or purple areas in the glands of the model group are significantly increased, indicating the presence of intestinal metaplasia. This condition is also improved after intervention with the traditional Chinese medicine composition provided by the present invention or FA, and the effect of the group treated with the traditional Chinese medicine composition provided by the present invention is more obvious ( Figure 6C and E). These findings indicate that both the traditional Chinese medicine composition provided by the present invention and FA can improve the pathological conditions of the gastric mucosa of rats induced by MNNG, and the improvement of the traditional Chinese medicine composition provided by the present invention is more significant. After investigating the potential correlation between cellular senescence and the therapeutic effect of the traditional Chinese medicine composition provided by the present invention on PLGC, the present invention conducted SA-β-Gal staining experiments on pathological sections. The results showed that the staining increased significantly in the gastric mucosa after MNNG induction. This staining intensity decreased significantly after intervention with the traditional Chinese medicine composition provided by the present invention or FA, and the traditional Chinese medicine composition provided by the present invention showed a more obvious improvement ( Figure 6 C and F). MUC2 and P16 are respectively used as classical biomarkers for PLGC and cellular senescence, and immunofluorescence double staining was used to observe their expression changes in different groups. The results showed that the expression of P16 was significantly up-regulated in the model group, and the expression trend of MUC2 was the same. After treatment with the traditional Chinese medicine composition provided by the present invention and FA, the expression levels of MUC2 and P16 decreased, and the traditional Chinese medicine composition provided by the present invention showed a more obvious effect ( Figure 6 C and G). In addition, cellular senescence is characterized by the expression of cyclins and SASP. The present invention first used RT-qPCR to evaluate the changes in the mRNA expression levels of cyclin p16, p21, p53 and IL-6, NF-κB and TNF in SASP. The results showed that the mRNA levels of these proteins were up-regulated in the model group and significantly down-regulated after intervention with the traditional Chinese medicine composition provided by the present invention or FA, and the traditional Chinese medicine composition provided by the present invention showed a more obvious effect ( Figure 6 H). The WB results also showed that the protein expression levels of p16, p21 and p53 were up-regulated in the model group and down-regulated after intervention with the traditional Chinese medicine composition provided by the present invention or FA ( Figure 6 I). These findings indicate that cellular senescence is related to PLGC, and the treatment with the traditional Chinese medicine composition provided by the present invention inhibits cellular senescence.

[0097] In vitro senolytic effect of the traditional Chinese medicine composition provided by the present invention on D-gal and MNNG-induced GSE-1 cells

[0098] To explore the relationship between cellular senescence and PLGC and the effect of the traditional Chinese medicine composition provided by the present invention on cellular senescence, the present invention used GSE-1 cells as a research model. The present invention established an in vitro PLGC model using MNNG-induced MC cells. After screening the MNNG concentration by CCK-8 experiment, the present invention determined that 30 μM / ml of MNNG was toxic, so 20 μM / ml was selected as the intervention concentration ( Figure 11 A). After induction, significant changes occurred in the cell morphology; compared with normal spindle-shaped GSE-1 cells, MC cells showed a polygonal and irregular shape ( Figure 11B). Subsequently, the present invention determined the intervention concentration of the traditional Chinese medicine composition provided by the present invention at 48 hours using 7.5% drug-containing serum, which was selected by CCK-8 ( Figure 11 C and 11D). D-galactose (D-gal), a commonly used reagent for inducing cell senescence, was used to treat GES-1 cells to observe the effect of the traditional Chinese medicine composition provided by the present invention on cell senescence. The present invention evaluated the intervention concentration of D-gal by detecting the expression level of p16 protein by Western blotting, and finally selected 40 mg / ml as the intervention concentration ( Figure 11 E and 11F).

[0099] Subsequently, the present invention first performed SA-β-Gal staining and observed green-stained cells to varying degrees after induction by D-gal and MNNG. After intervention with the traditional Chinese medicine composition provided by the present invention, the number of stained cells decreased ( Figure 7 A and B). Next, the present invention performed a cell scratch assay to evaluate cell migration. The results showed that after induction by D-gal and MNNG, the migration ability of MC cells was significantly enhanced, and similar trends were also shown in senescent GES-1 cells. After intervention with the traditional Chinese medicine composition provided by the present invention, the migration abilities of senescent GES-1 cells and MC cells were inhibited ( Figure 11 G and 11H). The present invention also measured cell viability, and the results showed that after induction by D-gal and MNNG, the proliferation of GES-1 cells was significantly inhibited, while intervention with the traditional Chinese medicine composition provided by the present invention was improved ( Figure 11 I). These findings indicate that MNNG-induced MC cells and D-gal-induced senescent cells showed similar trends in senescence biomarkers such as SA-β-Gal, migration, and proliferation, all of which could be improved by intervention with the traditional Chinese medicine composition provided by the present invention. Subsequently, the present invention further observed the molecular expression levels. Immunofluorescence of p16 showed that after induction by D-gal and MNNG, compared with normal GES-1 cells, the expression of p16 was upregulated in both groups and was inhibited after intervention with the traditional Chinese medicine composition provided by the present invention ( Figure 7 A and D). RT-qPCR results also showed that after induction by D-gal and MNNG, the mRNA expression levels of cell cycle proteins p16, p21, p53, and SASP-related genes IL-6, NF-κB, and TNF were upregulated and were improved after intervention with the traditional Chinese medicine composition provided by the present invention ( Figure 7 C). Western blotting results further showed that after induction by D-gal and MNNG, the protein expression levels of cell cycle-related proteins p16, p21, and p53 were upregulated and were also improved after intervention with the traditional Chinese medicine composition provided by the present invention ( Figure 7E and F). The above results indicate that MNNG-induced MC cells exhibit cellular senescence, and the traditional Chinese medicine composition provided by the present invention can eliminate senescent cells induced by D-gal and MNNG, as well as inhibit the expression of senescence-related proteins and SASP.

[0100] As can be seen from the above examples, the traditional Chinese medicine composition provided by the present invention not only treats PLGC, but also inhibits cellular senescence and the expression of cyclin and SASP. This therapy may become an important measure for the future treatment of PLGC.

[0101] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: In parts by weight, it contains the following raw materials: 13-17 parts of Artemisia capillaris, 10-14 parts of Scutellaria baicalensis, 13-17 parts of Hedyotis diffusa, 13-17 parts of Isatis indigotica, 13-17 parts of Lobelia, 7-11 parts of Patchouli, 13-17 parts of Scutellaria barbata, 8-12 parts of Sophora flavescens, 10-14 parts of Coptis chinensis, 13-17 parts of Gynostemma pentaphyllum, and 7-11 parts of Cyperus rotundus.

2. The Chinese medicine composition according to claim 1, characterized in that: The dosage form of the Chinese medicine composition is selected from tablets, pills, capsules, granules, powders, oral liquids, syrups, pastes or aerosols.

3. The Chinese medicine composition according to claim 1, characterized in that The Chinese medicine formula also includes pharmaceutically acceptable excipients.

4. Use of the Chinese medicine composition according to claim 1 in preparing a drug for treating gastric precancerous lesions.

5. The method for preparing the Chinese medicine composition according to claim 1, characterized in that: The following steps are involved: All raw materials are mixed with water, boiled and concentrated to obtain the traditional Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that: The weight ratio of the raw material drug to water is 1:5-10.

7. The preparation method according to claim 5, characterized in that: The concentration multiple of the concentration is 30 to 40 times.

8. The preparation method according to claim 5, characterized in that: The decoction includes boiling for 5 to 15 minutes and then simmering over a low heat.

9. Use of the traditional Chinese medicine composition according to any one of claims 1 to 3 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 8 in the preparation of a senescent cell elimination agent.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 3 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 8 in the preparation of an agent for regulating aging-related secretory phenotype.

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