Compound Kushen Injection Inhibits EMT Of Gastric Cancer Cells Through PI3K/AKT Pathway

BackgroundGastric cancer is a malignant tumor disease that originates in the human digestive tract.It is highly aggressive and is usually one of the important causes of death in patients with gastric tumors.Under normal circumstances,patients with gastric malignant tumors are diagnosed as middle and advanced stages,so the mortality rate of patients is relatively high.This is also one of the great challenges faced by existing medical related fields in seeking effective methods to deal with gastric malignant tumor diseases..As a serious threat to human life and health,malignant tumor diseases are easy to metastasize and easy to relapse,which is a huge challenge faced by current medical methods in the process of their diagnosis and treatment.More and more studies have confirmed that the Epithelial-mesenchymal transition(EMT)phenomenon plays a key role in the metastasis and recurrence of malignant tumors.The phenomenon of epithelial cell-mesenchymal transition usually refers to the transformation of cells from the epithelial phenotype of the organs and tissues of the body to the mesenchymal phenotype.It plays an important role in various types of physiological or pathological processes of the body,such as embryonic development,fiber and wound repair.Scientific research has shown that many types of cell pathway factors and key genes are involved in the process of EMT,including Snail protein genes,Slug protein genes and Twist protein genes and other transcription factors.In the process of epithelial cell-mesenchymal transition,the expression of epithelial phenotypic markers such as keratin and E-cadherin are weakened,and the expression of fibronectin and ?-catenin Significantly up-regulated expression of a variety of mesenchymal phenotypic markers dominated by ?-catenin,vimentin and N-cadherin.Scientific research has shown that both the classic Smad signal pathway and the non-classical signal transduction pathway play an important role in the process of EMT of the same malignant tumor cells.Studies have shown that transforming growth factor binds to E-type receptors in the Smad signaling pathway,which in turn activates Smad2 and Smad3,and then binds to Smad4.Finally transfer to the nucleus and play a regulatory role.The PI3K/AKT signaling pathway and the mitogen-activated protein kinase(MAPK)signaling pathway as non-classical signaling pathways have been shown to interact with classic pathways such as the Smad signaling pathway and promote the formation of EMT in gastric malignant tumors..Studies have shown that phosphatidylinositol-3-kinase(PI3K)can produce a specific catalytic effect on the third hydroxyl group of phosphatidylinositol(PI),and then the latter will be phosphorylated.Serine/threonine protein kinase(AKT),as a key target kinase downstream of PI3K,not only has the activity characteristics of serine,but also has the activity of threoninase.Insulin growth factor or survival factor can activate the signal pathway activity of serine/threonine protein kinase,which is composed of more than 480 amino acid residues.Studies have shown that PI3K not only possesses the active function of serine/threonine kinase,but also exerts the active effect of phosphatidylinositol kinase.According to the different structure and functional characteristics of PI3K,it can be roughly divided into three types,with different structures and functions.Among them,the most widely studied type I PI3K,as a heterodimer structure of PI3K,is mainly composed of one catalytic subunit and one regulatory subunit.The regulatory subunits also include two different domains,SH2 and SH3,which can interact with their corresponding specific binding site target proteins.Scientific research has confirmed that there are two main ways to activate the PI3K signal transduction pathway:the first is through the interaction with the connexin containing phosphorylated tyrosine residues,while causing conformational changes to the corresponding dimer.The activation can also interact with certain growth factor receptors to produce corresponding dimer conformational changes to achieve the activation effect;secondly,PI3K can increase its activity when combined with p110 and Ras,thereby activating PI3K signaling pathway.The PI3K/AKT signaling pathway is closely related to the spread,invasion and metastasis of cancer,and is one of the most frequent and critical signaling pathways in cancer.In recent years,more and more relevant research reports have confirmed that PI3K/AKT is the same as liver cancer,kidney cancer,bladder cancer,lung cancer,breast cancer,and gastric cancer.The diseases are closely related,and all participate in and affect the EMT process to varying degrees,but the EMT process of gastric cancer cells is rarely reported at present.In recent years,the natural medicine products provided by experts and scholars in the pharmaceutical industry have found that Chinese herbal medicine contains many ingredients,and different ingredients play different pharmacological effects.However,it is precisely because traditional Chinese medicine preparations contain too many active ingredients and the mechanism of action is complicated,making it difficult to study the mechanism of Chinese medicine on the human body.A gap between modern medicine and traditional Chinese medicine is that the current understanding and understanding of the mechanism of Chinese medicine is still relatively vague.Therefore,it is necessary to establish a traditional network pharmacology method to study Chinese medicine.The method of network pharmacology research is mainly based on the systemic and molecular level,discovering the active ingredients in traditional Chinese medicine,clarifying the mechanism of action of each active ingredient and predicting the target genes and Pharmacological effects.Through network pharmacology research,the therapeutic effect of drugs can be improved,and toxic and side effects can be reduced,thereby increasing the success rate of clinical trials of new drugs and saving drug research and development costs.In order to effectively integrate traditional Chinese medicine and modern medicine,researchers have established a pharmacology platform that integrates medicinal chemistry,pharmacokinetics,and drug-target-disease networks--the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP).The database contains 499 herbal medicines and the compound components of each herbal medicine,and provides comprehensive evaluation data of human absorption,distribution,and metabolism properties for each compound.It provides the targets of potential active molecules and their disease information,forms a drug-target-disease network for each herbal medicine,and provides a new platform for studying the mechanism of Chinese medicine at the system level.The network pharmacology of Chinese medicine is forced by the form and the general trend.Finding a model and method suitable for the research of the Chinese medicine system is the key to the modern development of Chinese medicine.Network pharmacology emphasizes the combination of biological network and drug action network,by analyzing the nodes or network modules in the network of drug active ingredients and disease-related genes,from finding a single ingredient to comprehensive network analysis,to further clarify the effectiveness of each drug anti-cancer mechanism of ingredientsCompound Kusen injection(CKI)is a mixture of natural compounds extracted from Kusen and Bai Tuling.It has been used to assist anti-cancer clinical treatment for more than 15 years.The main component is the herbal Kusen.Kusen contains a variety of pharmacologically active ingredients such as matrine and oxymatrine.It has a variety of pharmacological activities,including anti-inflammatory,anti-viral,anti-allergic and cardiovascular protection.Especially in terms of anti-cancer,it has been proved to have a significant effect on cancer or tumors.In addition,studies have confirmed that the 33 active ingredients contained in CKI are closely related to multiple anti-cancer signal pathways.Among them,quercetin has been shown to have a variety of biological effects,including inhibiting cell proliferation in various cancers and participating in anti-cancer signal transduction pathways[34].The signal that naringenin induces apoptosis is that it induces apoptosis mediated by reactive oxygen species(ROS)by regulating kinase 1(ASK1),which reveals the mechanism by which Kusen inhibits pancreatic cancer cells.There is also evidence that the luteolin contained in Kusen can inhibit the proliferation of colon cancer through the expression of miR-384 pleiotropic protein(PTN),and has a potential inhibitory effect on colorectal cancer cells.The glycerol contained in Kusen can not only reduce the expression of mitosis-related genes to exert anti-tumor effects37.Moreover,it can inhibit cancer cells by regulating the miR-21-mediated PTEN/AKT signaling pathway.[38]The active ingredient of naringenin in Kusen can inhibit the proliferation of prostate cancer cells and induce their apoptosis through the PI3K/AKT signaling pathway.In addition,the network pharmacology study of traditional Chinese medicine also found that the active ingredients contained in Kusen may induce cancer cell apoptosis through P53 and PI3K/AKT signaling pathway mechanisms.Based on the analysis of bioinformatics in the network pharmacology of traditional Chinese medicine,this paper fully discusses the various possible mechanisms by which CKI may affect the proliferation,differentiation,survival,and metastasis of gastric cancer cells.It also discusses the effects of Kushen injection on gastric cancer through PI3K/AKT pathway The regulation of the cell EMT process is verified,and explanations are given from two aspects of bioinformatics research and in vitro research,which will give some clinical inspiration.Chapter 1:Network Pharmacological Analysis of Traditional Chinese MedicineObjective:Through the network pharmacology of traditional Chinese medicine,various effective components contained in CKI and various genes and signal pathways related to gastric cancer are analyzed,and various possible mechanisms of CKI affecting the proliferation,differentiation,survival and metastasis of gastric cancer cells are discussed.Methods:Screen the effective drug components of Kushen and Bai Tuling in CKI from TCMSP.The screening criteria are Oral bioavailability(OB)>30%,drug-likeness(DL)>0.18.Based on the TCMSP database,the screening criteria are:After the effective drug components are targeted for collection.Based on GENECARD and OMIM databases,gastric cancer targets were collected.The Venn diagram is used to confirm the common target of effective drug ingredients and gastric cancer.Cytoscape software is used to establish a common target network of effective drug ingredients-gastric cancer,visualize processing,and perform topological analysis to predict the material basis of effective drug ingredients and key targets of drugs and diseases.Use the STRING online network to construct a protein-protein interaction network(PPI),and mine the core regulatory genes based on gene connectivity.GO(Gene Oncology)enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis were performed on common targets to predict the key pathway of CKI treatment of gastric cancer.Results:CKI has an effect on gastric cancer-related target genes through 35 effective drug ingredients.These 35 effective ingredients include kushenin,luteolin,Glyceollin,Phaseolin,quercetin,beta-sitosterol,etc.,and 162 genes are common targets for CKI and gastric cancer.The regulated core genes may include AKT1,IL6,MAPK1,JUN,VEGFA,EGF,MAPK3,EGFR,etc.Through GO enrichment analysis,the most enriched genes and the most significant function are DNA-binding transcription activator activity,RNA polymerase ?-specific,receptor ligand activity,cytokine receptor binding,cytokine activity,ubiquitin-like protein ligase binding,nuclear receptor activity,etc.Through KEGG enrichment analysis,the metabolic pathways with a higher degree of enrichment are PI3K-Akt signaling pathway,Human cytomegalovirus infection,Hepatitis B,Kaposi sarcoma-associated herpesvirus infection,Proteoglycans in cancer,AGE-RAGE signaling pathway in diabetic complications,etc.Pathway analysis shows that CKI may pass through the ERBB signaling pathway,PI3K/Akt signaling pathway,MAPK signaling pathway,AMP signaling pathway It regulates the growth,proliferation,apoptosis and invasion of gastric cancer cells.And CKI is likely to regulate the growth,proliferation,apoptosis,and invasion of gastric cancer cells by regulating the P53 gene.Conclusions:AKT1 is one of the key genes for CKI in the treatment of gastric cancer,and CKI may regulate the growth,proliferation,apoptosis and invasion of gastric cancer cells through PI3K/AKT signaling pathway and inhibit EMT of gastric cancer cells.Cell experiments are needed for further verification.Chapter 2:CKI inhibits EMT of gastric cancer cells through PI3K/AKT pathwayObjective:On the basis of network pharmacology analysis and research of Chinese medicine,fully explore the role of CKI in classical cell signaling pathways The role of PI3K/AKT and its effective inhibition of gastric cancer cell epithelial interstitial regulation.Methods:Select gastric cancer BGC803 and MKN28 cell lines for cell culture,select the first generation of monolayer cells as the research object;culture the cells overnight before treatment,set different concentration gradient CKI solutions for grouping during treatment,H-CKI(high concentration group,100?l CKI Injection/ml medium),L-CKI(low concentration group,20?l CKI injection and 80?l saline/ml medium),and set the control group(100?l CKI normal saline/ml medium),continue cell culture after CKI treatment After 48 hours,carry out relevant project analysis.The details are as follows:CCK-8 detects cell proliferation in each group;Scratch and Transwell test detects the invasion and migration ability of gastric cancer cells in each group;Western-blot detects E-cadherin,N-cadherin,Vimentin and PI3K markers of gastric cancer cells in each group PI3K/AKT signaling pathway key protein P13K and p-AKT1 transcription factor protein expression;flow cytometry to detect the apoptosis of gastric cancer cells in each group;flow cytometry to detect the cycle distribution of gastric cancer cells in each group,statistical data,and analysis.Results:1.CCK-8 detection of cell proliferation in each group:under the stimulation of CKI,the OD value of the two GC cells at 450 nm decreased significantly,and with the increase of CKI concentration,the two GC cells at 450 nm The OD value decreased more significantly,indicating that CKI can affect the proliferation rate of two GC cells,and this effect increases with the increase of CKI concentration.The comparison result of cell growth rate in each group was H-CKI<L-CKI<Control,data comparison between each group,P<0.05,the difference was statistically significant.2.Scratch test:CKI can significantly reduce the migration rate of gastric cancer cells.As the concentration of CKI increases,the migration orbit gradually widens,and the orbit width and CKI concentration show a negative correlation;the migration rate of each group is H-CKI<L-CKI<Control,data comparison between groups,P<0.05,the difference is statistically significant.Transwell invasion experiment:CKI can significantly reduce the invasion ability of gastric cancer cells.As the concentration of CKI increases,the invasion rate of gastric cancer cells gradually decreases,and the density of gastric cancer cells in the chamber gradually increases.The invasion comparison between each group is H-CKI<L-CKI<Control,data comparison between groups,P<0.05,the difference is statistically significant.3.Western-blot:With the increase of CKI concentration,the expression concentration of E-cadherin in gastric cancer cells of each group gradually increased,and the expression concentration of N-cadherin and Vimentin gradually decreased,reflecting the reduction of EMT degree;The expression trend of E-cadherin concentration is Control<L-CKI<H-CKI,the comparison of datas between each group,P<0.05,the difference is statistically significant;in the analysis of PI3K/AKT,as the concentration of CKI increases,PI3K and The p-AKT1 expression concentration gradually decreased,reflecting the negative regulation effect on the PI3K/AKT pathway;the concentration expression trend between each group was H-CKI<L-CKI<Control,the comparison of data between each group,P<0.05,the difference was statistical significant.4.Flow cytometry:As the increases of concentration of CKI,the apoptotic rate of gastric cancer cells in each group gradually increases.The comparison of the apoptotic rate between each group is H-CKI>L-CKI>Control,comparison of data between each group,P<0.05,the difference is statistically significant.With the increase of CKI concentration,the proliferation rate of gastric cancer cells in each group gradually decreased,and the G2/M ratio of gastric cancer cells in each group gradually increased.The comparison of the G2/M ratio between the groups was H-CKI>L-CKI>Control,Comparison of data between each group,P<0.05,the difference is statistically significant.Conclusions:1.CKI intervention can affect the proliferation rate of gastric cancer cells.With the increase of CKI concentration,the proliferation rate of gastric cancer cells shows a significant downward trend,and this effect increases with the increase of CKI concentration.2.CKI can significantly reduce the migration and invasion ability of gastric cancer cells.As the concentration of CKI increases,the migration rate and invasion rate of gastric cancer cells are significantly reduced,which is negatively correlated with the concentration of CKI.3.CKI can significantly increase the expression activity of E-cadherin and reduce the expression activity of N-cadherin and Vimentin,showing a dose-dependent characteristic.At the same time,CKI can regulate the expression of the key transcription protein activity of PI3K/AKT in the classical cell signaling pathway.As the concentration of CKI increases,the activities of the transcription proteins PI3K and p-AKT1 are significantly reduced,which shows that CKI can affect the EMT process of gastric cancer cells through the PI3K/AKT signaling pathway.