| Objective:To explore the effect of Sichong Tablet on the proliferation,apoptosis,migration and invasion of gastric cancer cells and related mechanisms,to observe the clinical efficacy of Sichong Tablet on patients with combination of blood stasis and toxin postoperative gastric cancer,and analyze the potential molecular biological mechanism of Sichong Tablet in the treatment of gastric cancer by network pharmacological methods.Methods:1.Experimental study: The CCK-8 method was used to detect the effect of different concentrations(0?g / ml?500?g / ml)of Sichong Tablet on the proliferation of human gastric cancer AGS and MKN45 cells,and calculate the proliferation inhibition rate and the IC50 value;The appropriate concentration of Sichong Tablet was selected,and the effect of Sichong Tablet on the proliferation of gastric cancer cells was detected by CCK-8 method and clone formation experiment,flow cytometry was used to detect the apoptosis rate,scratch test and Transwell chamber method were used to detect the cell migration and invasion ability.Western blot was used to detect the effect of Sichong Tablet on the expression of apoptosis-related proteins Bax,Bcl2,C-Caspase 3,?-tubulin and AKT /Cyclin D1 cell signaling,the effect of Sichong Tablet on MMP9 activity was detected using gelatin zymography.2.Clinical study: 64 patients with combination of blood stasis and toxin postoperative gastric cancer were randomly divided into a treatment group and a control group.The control group was given OLF chemotherapy.Based on this,the treatment group was given Sichong Tablet.Observe the differences in TCM symptom scores,KPS scores,lymphocyte subsets,tumor marker levels,circulating tumor cell(CTC)numbers,and adverse reactions to chemotherapy in the two groups for three chemotherapy cycles.Patients were followed up to observe the difference in Disease-free survival(DFS),research results were collected and performed statistical analysis.3.Network pharmacology study: all chemical components of Scorpion?Scolopendra?Eupolyphaga?Earthworm were retrieved from the CNKI,CBM,CQVIP,Wanfang database,Pub Med,Coremine database,all targets corresponding to the chemical components were retrieved through CNKI,CBM,TCMSP,TCM Database @ Taiwan,CTD;Targets related to gastric cancer were retrieved through CNKI,CBM,Pub Med,Gene Cards,OMIM databases,selected targets that interact with Sichong Tablet,Cytoscape was used to build the component-target-disease network,the String database was used to build the protein-protein interaction network,the Cyto NCA plug-in was used to perform network topology analysis on the core target,and the R package was used to enrich the core target function(GO),and the Clue GO plug-in was used to perform KEGG pathway enrichment analysis.Results:1.Experimental study: With the increase of the concentration of Sichong Tablet,the proliferation inhibition rate of gastric cancer AGS and MKN45 cells gradually increased.At 48 hours,the IC50 values of AGS and MKN45 cells were 240 ?g/ml and 200?g/ml.The study selected 80?g / ml as the drug concentration of the Sichong Tablet group for subsequent experiments;In the cell proliferation experiment,the level of inhibition of AGS and MKN45 cells in the Sichong Tablet group at 48 h and 72 h was significantly higher than that of the control group(P <0.05),which was time-dependent.At the same time,Sichong Tablet significantly reduced the colony forming ability of gastric cancer AGS and MKN45cells(P <0.05);In the study of apoptosis,the percentages of AGS and MKN45 cells in the Sichong Tablet group were(23.44 ± 1.2)% and(24.76 ± 2.1)%,which were significantly higher than those in the control group(4.03 ± 0.23)% and(4.63 ± 0.31)%;Compared with the control group,the expression of apoptosis-related proteins Bcl2,C-Caspase 3 in AGS and MKN45 cells in the Sichong Tablet group were significantly reduced,and Bax expression and Bax/Bcl2 ratio were significantly increased(P <0.05);In the scratch test,the percentage of healing area of gastric cancer AGS and MKN45 cells in the Sichong Tablet group were significantly lower than that in the control group(P <0.05);In the Transwell experiment,the migration and invasion of gastric cancer AGS and MKN45 cells in the Sichong Tablet group were significantly less than the control group(P <0.05),and the activity of migration and invasion related protein MMP9 was significantly lower than that of the control group(P <0.05);Compared with the control group,the Sichong Tablet group significantly reduced the AKT phosphorylation level of AGS and MKN45 cells and the expression level of the downstream effector Cyclin D1.2.Clinical study: A total of 61 patients were evaluated effectively,30 in the treatment group and 31 in the control group.In terms of TCM symptom scores,the total score of the treatment group after treatment was significantly lower than that of the control group.Among them,6 cases were markedly effective and 19 cases were effective in the treatment group,4 cases were markedly effective and 12 cases were effective in the control group,the effective rate of the treatment group was significantly higher than that of the control group(P <0.05);KPS in the treatment group improved in 19 cases,stable in 6 cases,KPS in the control group improved in 10 cases,stable in 13 cases.The KPS improvement in the treatment group was significantly better than that in the control group(P <0.05);The proportion of CD4+ and NK cells and the value of CD4+/CD8+ in the treatment group were significantly higher than those in the control group after treatment(P <0.05),and the levels of CEA,CA19-9,CA125,and CA72-4 were significantly lower than those in the control group after treatment(P <0.05);There was no significant difference in the number of CTCs between the two groups(P> 0.05);The number of adverse reactions to chemotherapy in the treatment group was significantly less than that in the control group(P<0.05);During the follow-up period,the DFS time in the treatment group was(15.33 ±5.78)months,and the DFS time in the control group was(12.48 ± 4.97)months.The treatment group was significantly longer than the control group(P <0.05).3.Network pharmacology study: In the study,200 chemical components of Sichong Tablet and 499 potential targets were retrieved.The active components in Sichong Tablet were mainly Adenosine,4,7,10,13,16,19-docosahexaenoic acid,Uridine,alanine,Bm K M1 neurotoxin,Phenol,5,8,11,14-Eicosatetraenamide,Batyl alcohol,hypoxanthine,Dihydrocapsaicin,etc.which can exert the effect of treating gastric cancer;A total of 1227 targets related to gastric cancer were retrieved and screened from the disease database,and155 common targets were obtained from the intersection.152 core targets were selected using network topology analysis,mainly involving NTRK1,TP53,EGFR,CUL3,XPO1,ESR1,MCM2,UBC,FN1,HSP90AA1,etc.157 functional areas were obtained by go enrichment analysis,among which 28 functions had more than 10 enriched genes,including cell adhesion,mRNA transcription,DNA binding,protein synthesis and transport,etc.KEGG enriched 75 signal pathways,39 of which had more than 10 enriched genes,it mainly involved in the Pathways in cancer?Viral carcinogenesis?Transcriptional misregulation in cancer?Cell cycle?PI3K-Akt signaling pathway?Ubiquitin mediated proteolysis,etc.Conclusion:1.(1)Sichong Tablet can significantly inhibit the proliferation of human gastric cancer AGS and MKN45 cells,and is related to its concentration and time.(2)Sichong Tablet can induce apoptosis of human gastric cancer AGS and MKN45 cells.The possible mechanism is through down-regulating the expression of apoptosis-related proteins Bcl2 and up-regulating the expression of Bax and C-Caspase 3.(3)Sichong Tablet can inhibit the migration and invasion of human gastric cancer AGS and MKN45 cells,and its mechanism may be related to the inhibition of the activity of MMP9.(4)Sichong Tablet can cause a decrease in AKT phosphorylation level and a decrease in the expression of its downstream effector Cyclin D1,and may affect the biological behavior of gastric cancer cells through this mechanism.2.Within 3 months of treatment cycle,Sichong Tablet can significantly improve the symptoms of TCM,quality of life,and lymphocyte subsets,and significantly reduce tumor marker levels and the incidence of adverse reactions to chemotherapy in patients.During the follow-up period,patients' DFS can be significantly extended.3.Sichong Tablet exerts its therapeutic effect on gastric cancer through multi-targets,multi-paths,and mutual coordination.The main active targets exert anti-tumor functions by interfering with cell adhesion,mRNA transcription,DNA binding,protein synthesis and transport,it also plays a role by affecting cell cycle-like signal pathways,mainly involving Pathways in cancer,Viral carcinogenesis,Transcriptional misregulation in cancer,Cell cycle,PI3K-Akt signaling pathway,Ubiquitin mediated proteolysis,Hepatitis B,Alcoholism,Human papillomavirus infection,MAPK signaling pathway,Hepatitis C,Micro RNAs in cancer,Proteoglycans in cancer,these therapeutic effects fully reflect the overall and systemic characteristics of Sichong Tablet in the treatment of gastric cancer... |
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