| BackgroundTriple negative breast cancer(TNBC)is a breast cancer with negative expression of estrogen receptor,progesterone receptor,and human epidermal growth factor receptor,witch accounts for 15-20%of all breast cancers.TNBC has the characteristics of high regional recurrence rate,high distant metastasis rate,and poor prognosis after treatment.Distant metastasis after treatment is the leading cause of death for patients.Patients newly diagnosed with TNBC generally often recur within three years.The 5-year relative survival rate of patients with triple-negative breast cancer with distant metastasis is 11.5%.Surgery and chemotherapy are still the main treatments for triple-negative breast cancer.Currently,there are no specific targeted therapies,so Chinese medicine could play a important role in the replacement treatment of TNBC and the therapeutic effect of Chinese medicine is significance to improve the quality of life of patients,prolong the recurrence-free survival period of patients,so it is meaningful explore its intrinsic mechanism of function.Breast cancer belongs to the category of "Ruyan" in Chinese medicine.Insufficient "Qi" is the fundamental pathogenesis of tumor recurrence.Risky factors hide in the body,and waiting for opportunity to harm the body is the direct factor of tumor recurrence.Insufficient endowments,internal injuries of the emotions,and tiredness caused by eating and drinking are all factors that cause tumors,and modern medical treatment methods such as surgery and chemotherapy can easily hurt the "Qi",and if there is cancer remaining on the premise of deficiency "Qi" can't control risk factors,those factors are more likely to recur or flow elsewhere,leading to tumor recurrence and metastasis."Qi" deficiency provides a prerequisite for tumor recurrence and metastasis.On this basis,all the threatening factors are mutually associated with each other,and then become cancerous,causing tumors to recur and metastasize.A number of studies have shown that Chinese medicine treatment for tumor patients can extend the relapse-free survival of patients by adjusting the balance of yin and yang,qi and blood,and organs of the body,and even achieve the effect of"preventing disease and preventing change".Professor Lu summed up "Sanyin"Recipe based on many years of clinical experience in treating TNBC.On this basis,the patient's tongue,pulse,discomfort and physical differentiation are usually combined with addition and subtraction.In the cohort study conducted from January 2007 to May 2012,we randomly divided 101 patients with triple negative breast cancer into a cohort of syndrome differentiation taking traditional Chinese medicine combined with Western medicine treatment and a pure western medicine treatment group.Progressive survival and patient quality of life are observed.The results showed that the 3-year progression-free survival of the integrated Chinese and western medicine group showed certain advantages over the simple western medicine treatment group.Chinese medicine can improve the symptoms of numbness and chemotherapy brain caused by chemotherapy,and improve the quality of life.In addition,we also participated in the Beijing Municipal Science and Technology Commission's Intervention Study on the Recurrence and Metastasis of Postoperative Recurrence and Metastasis in Patients with Triple Negative Breast Cancer by the Beijing Science and Technology Commission.In the group of 60 patients with intermediate and advanced triple-negative breast cancer,the 3-year recurrence-free survival rate of Chinese medicine treatment was 88.3%,and according to SEER,the 3-year recurrence-free survival rate of triple-negative breast cancer was 66%.Its internal mechanism are worthy of further study.Network pharmacology is an efficient method for studying the medicinal effect mechanism of traditional Chinese medicine compound prescriptions.By constructing a visual network,it can provide data information on drug active ingredients,action targets,and related signal pathways from multiple levels,so as to predict the core components and Mechanism.Therefore,this research is divided into two parts.The first part explores the core medicine of Sanyin formula through network pharmacology and predicts the possible mechanism of action of Sanyin formula.The second part conducts experimental research to verify the internal mechanism of this formula based on the data analysis results of Part I.Purpose:Through the network pharmacology method,optimize the compatibility of Sanyin prescription,explore its core action components and internal mechanism of action,and combine the in vitro experiments to observe the biological characteristics of Sanyin prescription on breast cancer cell proliferation,metastasis,and invasion.To explore the effect of Sanyin formula on breast cancer metastasis based on the network pharmacological prediction mechanism.Method:Part 1:Online Pharmacology(1)Search the network database to find the effective ingredients and targets of Sanyin formula and summarize them.By searching the "GeneCard" database,target genes related to triple negative breast cancer were obtained.The target genes of triple-negative breast cancer and target proteins of active ingredients of traditional Chinese medicine were molecularly docked,and a "drug-disease-target" network was established using Cytoscape software.(2)Analyze the "drug-disease-target" network data of Sanyin formula,summarize the core medicine and active ingredients of Sanyin Decoration,and optimize the compatibility of Sanyin formula.Through the establishment of Sanyin formula's PPI network,GO biological function enrichment analysis and KEGG signal pathway enrichment analysis were performed to screen Sanyin formula's possible targets,biological functions,and related signal pathways.Part ?:Experimental Research(1)The optimized Sanyin recipe was used as the research object to prepare drug-containing serum.A blank serum group was set as a negative control group.High-,medium-,and low-concentration drug-containing serum were added to culture TNBC cells for 24,48,and 72hours.Then,using the method of CCK8 experiment,observe the effect of intervention drugs on breast cancer cell proliferation ability,find out the optimal concentration group and the optimal intervention time.(2)Select TNBC cells and measure the effect of intervention drugs on apoptosis of Sanyin breast cancer cells by flow cytometry according to AV-PI method.(3)The intervention drug with different concentrations was added to culture the triple negative breast cancer cells,and the effect of the intervention drug on the migration capacity of triple negative breast cancer cells was observed by the Transwell migration experiment.(4)Culture Sanyin breast cancer cells,add different concentrations of intervention drugs,and observe the effect of intervention drugs on the invasion ability of Sanyin breast cancer cells by Transwell invasion experiment.(5)Through RT-PCR experimental methods,the results of research and analysis based on network pharmacology were examined to explore the core mechanism of action of Sanyin formula.Result:Part ?:Online Pharmacology(1)41 active ingredients in Sanyin Recipe were retrieved and 3885 target genes related to Sanyin breast cancer were obtained.On this basis,duplicate and unrelated terms were deleted to obtain Sanyin Recipe and TNBC-associated active ingredients.32 The total number of targets was 159,and the "drug-disease-target" network of Sanyinfang was constructed.(2)According to data analysis,the action network of astragalus,saponin,and Hedyotis diffusa in Sanyinfang is extensive,and it is the core medicinal taste in Sanyinfang;quercetin,kaempferol,stigmasterol,P-gluten Sterols are the main active ingredients in Sanyin Recipe;AKT1,IL6,VEGFA,CASP3,JUN,EGFR,EGF,etc.are located at the core of the PPI network;from the analysis of GO biological function analysis and KEGG signal pathway enrichment analysis,Sanyin Recipe The biological functions involved in the target protein mainly include transcription regulation,cell proliferation and apoptosis,etc.The PI3K/AKT signaling pathway is the main pathway involved.It is speculated that Sanyin Fang may inhibit cell proliferation and promote tumor cell apoptosis by acting on signal pathways such as PI3K/AKT.epithelial-mesenchymal transition Transition(EMT)is the central axis of tumor metastasis.Tumor cells gain strong invasion through EMT.During this process,E-cadherin is down-regulated and cell-cell adhesion is weakened.Vimentin and N-cadherin are up-regulated,and epithelial cells lose cell polarity and connection with the basement membrane.Changes in the three are important indicators of the occurrence of EMT transformation.Studies have shown that the PI3K/AKT signaling pathway is closely related to the EMT transformation process,so it is speculated that Sanyin formula may interfere with tumor cell EMT through the PI3K/AKT signaling pathway,thereby inhibiting tumor cell migration and invasion.Part ?:Experimental Research(1)The effect of intervention drugs on the proliferation of breast cancer cells was observed by the CCK8 experiment.After adding different concentrations of drug-containing serum,the results showed that the high-concentration group intervened for 24 hours and 48 hours,and the medium-concentration group intervened for 48 hours.The OD value of the cells was lower than In the control group,the results were statistically significant(P<0.05),indicating that the high-concentration group intervened for 24h and 48h,and the medium-concentration group intervened for 48h,and the cell proliferation ability was inhibited.The high-concentration group had the most obvious inhibitory effect on serum for 24h.The rate was 38.7%,and the inhibitory effect of the optimized Sanyin prescription drug-containing serum on the proliferation of 4T1 cells was concentration-dependent,but not time-dependent.(2)In the AV-PI experiment,the apoptosis rate of cells treated with high-concentration intervention drugs for 24 hours was 18.33%.Compared with the Control group,the apoptosis rate of the intervention group increased,and the results were statistically significant(P<0.05),Indicating that high concentration of drug-containing serum can promote the apoptosis of 4T1 cells.(3)Transwell cell test was used to test the cell migration ability.The results showed that the drug-containing serum in the high and medium concentration groups could inhibit cell migration(P<0.05),and the effect of the high concentration group was better than that of the medium concentration group(P<0.05).The low concentration of drug-containing serum had no significant inhibitory effect on cell migration ability(P>0.05),indicating that the inhibitory effect of Sanyin fang drug-containing serum on cell migration ability was concentration-dependent.(4)Transwell chamber experiments were used to detect the invasion of cells.The results showed that the high and medium concentration drug-containing serum could inhibit the cell invasion,respectively(P<0.05),but the high and medium concentration drug-containing serum affected cells.There was no significant difference in the invasion effect(P>0.05).There was no significant inhibition of cell invasion in the low-concentration group(P>0.05).There was no significant concentration-dependent inhibition effect of Sanyin fang on cell invasion..(5)Through RT-PCR experiments,it was found that the expression of E-cadherin in cells increased after intervention with Sanyin formula's core drug-containing serum(P<0.05),and the relative expression of Vimentin decreased(P<0.05),N-cadherin expression did not change significantly(P>0.05),but showed a downward trend,and the cell EMT process was inhibited.conclusion:(1)According to the analysis results of network pharmacological data,the optimized prescription of Sanyin Fang is:Astragalus,Saponin,Hedyotis diffusa,the main active ingredients are quercetin,kaempferol,etc.,and the main targets are AKT1,IL6,VEGFA,CASP3,JUN,EGFR,EGF,PI3K/AKT and other signaling pathways may be involved.The possible mechanism of Sanyin prescription is to inhibit cell proliferation and promote tumor cell apoptosis.It may interfere with tumors through the PI3K/AKT signaling pathway.EMT of cells,which inhibits cell migration and invasion.(2)The experimental part basically conforms to the prediction mechanism of network pharmacology.Optimizing the Sanyinfang drug-containing serum can effectively inhibit cell proliferation,induce apoptosis,inhibit cell migration and invasion ability,and inhibit epithelial-mesenchymal transition in Sanyin breast cancer cells.. |
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