| Background:Gastric cancer(GC)is one of the most common malignant tumors in the world,posing a serious threat to human health.Modern studies show that all kinds of chemical components contained in Chinese herbal medicine have anti-tumor effects.Through excavating and analyzing TCM compounds involved in clinical practice,appropriate TCM compounds were selected for in-depth study to explore and analyze the molecular mechanism of its anti-gastric cancer,thus providing theoretical basis for clinical treatment of gastric cancer and reference for the development of new drugs to treat gastric cancer.Objective:To explore the law of prescription composition in treating gastric cancer,analyze the value of heat-clearing and detoxifying Chinese medicine in the treatment of gastric cancer,and select the target Chinese medicine.The pharmacological effects of the volatile oil components of Rabdosia rubescens(R.rubescens)(the target Chinese medicine)on gastric cancer were preliminarily studied by using network pharmacology analysis method combined with tumor database mining technology,and then preliminarily verified by molecular biology experiments,providing theoretical support for the molecular mechanism of R.rubescens in the therapy of gastric cancer.(1)Screening the key targets of the essential components of R.rubescens in the treatment of gastric cancer,and constructing a "TCM-compound-target-disease" regulatory network;(2)To verify the expression of key targets in gastric cancer tissues and explore the molecular mechanism of R.rubescens in the treatment of gastric cancer.Methods:(1)By sorting out TCM prescriptions involved in the clinical treatment of gastric cancer in recent years,a database was established and the statistics of frequency of TCM categories of four qi and five herbs were conducted.Cluster analysis and factor analysis were performed by IBM SPSS Statistics 21.Analysis of common clinical heat-clearing and detoxification Chinese medicines,screening target Chinese medicines,clinical value analysis.(2)DEGs between gastric cancer tissue and normal tissue was screened by analyzing two gastric cancer related data sets in GEO database.GO and KEGG enrichment were performed on DEGs by Metascape tool.At the same time,String database and Cytoscape software were used to construct the interaction network of DEGs,and the differential genes with high score in the interaction network were screened,so as to further explore the occurrence and development of gastric cancer.The GEPIA online tool was used to analyze the correlation between the above-mentioned differential genes and the survival of gastric cancer patients,and identify the key genes in gastric cancer.(3)Based on the research strategy of network pharmacology,the potential targets of essential oil active components of R.rubescens were predicted using resource platforms such as published studies,TCMSP,Swisstar Prediction and Pharmapper.The key gene set of gastric cancer analyzed in Chapter 2 was used as the disease target to predict the network regulation mechanism of the R.rubescens volatile oil in the treatment of gastric cancer through the protein interaction network.(4)To further verify the intervention mechanism found in network pharmacology study,the expression levels of TNF-α,IL-1β,MMP9 and PTGS2 proteins in gastric cancer and adjacent tissues were determined by IHC method.Next,gastric cancer cells were treated with volatile oil of R.rubescens at different concentrations to explore the effects of volatile oil on gastric cancer cell growth and downstream targets protein and mRNA expression levels,and then to explore the possible mechanism of volatile components in the development of gastric cancer.Results:(1)Among the 128 prescriptions included in the analysis,260 kinds of Traditional Chinese medicines were involved,and the top 5 Chinese medicines with 1153 times of use frequency were Atractylodes macrocephala,Hedyotis diffusa,Astragalus membranaceus,Poria cocos,Pinellia ternata.The top 5 Chinese medicines in terms of frequency were tonifying qi drugs,clearing heat and detoxifying drugs,improving water permeability,removing blood stasis,regulating qi drugs.In the treatment of gastric cancer,warm drugs(257 times,37.62%)and cold drugs(242 times,35.43%)were the most frequently used,while sweet drugs(329 times,48.17%)were the most frequently used,and the distribution of drugs in the three channels of spleen,lung and stomach was the most frequent.Cluster analysis classified the above-mentioned high-frequency Chinese medicines into 10 categories.Factor analysis showed that 8 common factors could be extracted,with a cumulative contribution rate of 66.40%.High frequency antipyretic and detoxifying drugs include:Hedyotis diffusa,Scutellaria barbata,R.rubescens and the root of Rattan pear,Professor Liu Shangyi prescribed 248 prescriptions,R.rubescens was used 210 times,ranking third.Professor Liu qiquan’s prescriptions involved 155 Chinese herbs,among which R.rubescens was used 70 times,ranking first in frequency.R.rubescens was selected as the target Chinese medicine for follow-up study.(2)Differential genes of gastric cancer were screened from GEO database,and 751 differential genes of gastric cancer were obtained.GO and KEGG enrichment analysis were performed for differential genes of gastric cancer.The characteristics of differential genes related to the occurrence and development of gastric cancer were analyzed from the aspects of molecular functions,biological processes,cell components and signaling pathways.The PPI network of 751 differential genes was successfully constructed,and the TOP50 differential genes were screened out,Survival analysis showed that COL1A1 and TIMP3 were significantly correlated with overall survival(OS),while only TIMP3 was correlated with disease free survival(DFS).(3)Thirteen volatile components of R.rubescens were selected as pharmacologically active components.The 13 active components and gastric cancer have a total of 83 targets,and a Traditional Chinese Medicine-component-targets-gastric cancer network was successfully constructed.Four core targets were obtained:TNF-α,IL-1β,MMP9,and PTGS2.The core components were oleic acid,palmitoleic acid,peach aldehyde,9-decenoic acid and 3,5-di-tert-butylphenol.(4)The expression levels of TNF-α,IL-1β,MMP9 and PTGS2 in gastric cancer tissues were significantly higher than those in adjacent tissues.CCK-8 results showed that the cell inhibition rate was significantly increased after the gastric cancer cell lines MKN-45 and HGC-27 were stimulated with different concentrations of R.rubescens volatile oil,and the activity of gastric cancer cells was inhibited in a concentration-dependent manner.Flow cytometry showed that the apoptosis rate of volatile oil in low dose and high dose groups was significantly higher than that in control group,and the apoptosis rate of volatile oil in high dose group was significantly higher than that in low dose group.Western Blot and RT-PCR showed that the protein and mRNA expression levels of TNF-α,IL-1β,MMP9 and PTGS2 in the low dose and high dose groups were significantly lower than those in the control group,and the high dose group was significantly lower than that in the low dose group.Conclusions:There are many volatile compounds in R.rubescens regulate gastric cancer.Network pharmacology and bioinformatics analysis indicated that the core targets of the volatile oil of R.rubescens on gastric cancer might be TNF-α,IL-1β,MMP9 and PTGS2 The expressions of TNF-α,IL-1β,MMP9 and PTGS2 proteins in gastric cancer tissues were significantly higher than those in adjacent tissues.The volatile oil of R.rubescens could effectively inhibit the proliferation and apoptosis of MKN-45 and HGC-27 cells,and inhibit the protein and mRNA expression of TNF-α,IL-1β,MMP9 and PTGS2. |
