| BACKGROUNDThe mortality of ovarian cancer ranks the first among gynecological malignant tumors.Due to its insidious onset,75%of the patients were in advanced stage at the time of diagnosis.Surgery combined with chemotherapy is the standard treatment,but the survival rate of advanced ovarian cancer has not been significantly improved in the past 30 years,and the 5-year survival rate of advanced patients is still less than 30%.Late maintenance treatment can improve the patients survival and quality of life and standard maintenance therapy after treatment with what method is the challenge for the medical workers,with modern molecular biology technology and the development of human genomics,precision become the trend of cancer treatment,precise targeted therapy drugs now angiogenesis inhibitors and PARP inhibitors have maintenance treatment for ovarian cancer,and showed a certain curative effect,but by reason of expensive and associated with side effects limit its use in clinical.To explore new maintenance therapy for ovarian cancer is an urgent clinical problem.Traditional Chinese medicine maintenance treatment of ovarian cancer is widely used in clinical,effective.YHJD is an effective prescription for maintenance treatment of advanced ovarian cancer based on the addition and reduction of "Lichong Decoction"by zhang Xichun,a modern famous doctor.Preliminary clinical trials have shown that,compared with the placebo group,maintenance treatment of YHJD can prolong the median progress-free survival period of patients with advanced ovarian cancer,improve the quality of life of patients and TCM symptoms.It can down-regulate peripheral blood regulatory T cells and up-regulate cytotoxic T cells.Animal experiments showed that YHJD inhibited the expression of immunosuppressive factors TGF-1 and IL-10 related to Tregs.Cell experiments showed that YHJD could inhibit angiogenesis,which was related to the effect of this prescription on Trees to promote tumor angiogenesis.A comprehensive analysis of clinical and basic studies shows that YHJD maintenance therapy can prolong the survival of patients with advanced ovarian cancer,which may play an anti-tumor role through multiple targets.There are many active components of YHJD,while ovarian cancer is a heterogeneous and complex disease with abnormal multi-gene and multi-pathway.Therefore,it is necessary to clarify the effective mechanism by comprehensively studying the interaction between the active components of YHJD and the multi-target and multi-pathway of the disease.Through the analysis of the two groups with the most significant difference in efficacy after the treatment of YHJD,the mechanism of the difference in efficacy was explored from the perspective of molecular biology,so as to clarify the genetic characteristics of the advantaged population in the treatment of YHJD.Network pharmacology emphasizes the use of network method to study the overall dynamic nature of multi-target drugs acting on human body,and has been widely used in the research of the core effective components,internal mechanism,intervention targets and systemic mechanism of TCM in the treatment of complex diseases.Ovarian cancer has different molecular subtypes and genomic characteristics,and studying the genetic characteristics in patients’ blood may reveal predictors of therapeutic efficacy.Therefore,this study intends to use network pharmacology and genomics research methods.The core Chinese medicine,core active component,target,pathway and action mechanism of YHJD for ovarian cancer were studied from the perspective of network pharmacology.In vivo animal experiments verified the anti-tumor effect of the core Chinese medicine obtained from the network pharmacology analysis on ovarian cancer tumor-bearing mice;From the perspective of mRNA chip combined with network pharmacology,mRNA chip will be applied to the two groups of patients with the longest and shortest survival time without progression after taking YHJD,to obtain differential gene combined with network pharmacology related to differential efficacy,and further study the target,pathway and molecular mechanism of differential efficacy of YHJD,so as to provide certain reference for clinical precision therapy of YHJD.PURPOSES(1)To explore the core TCM,core active components,targets,pathways and action mechanism of YHJD 17 in the treatment of ovarian cancer from the perspective of network pharmacology;(2)To construct a mouse model of ovarian cancer and verify the reliability of the network pharmacological results by observing the tumor suppressive experiment of YHJD core Chinese medicine in treating transplanted tumors in tumor-bearing mice;(3)From the perspective of clinical mRNA chip combined with network pharmacology,the target,pathway and molecular mechanism related to the differential efficacy of YHJD after clinical treatment were discussed,so as to provide certain reference for clinical precision therapy of YHJD.METHODSThe research is divided into three parts:1.Network pharmacology research on the action mechanism of YHJD in ovarian cancer;2.Experimental study on the antitumor effect of YHJD on ID8 bearing mice transplanted with tumor;3.MRNA microarray combined with network pharmacology for differential efficacy mechanism of YHJD in ovarian cancer.The details are as follows:I.Network pharmacology study on the action mechanism of YHJD in the treatment of ovarian cancer(1)Data acquisition using TCMSP,ETCM,BATMAN-TCM and TCMID database input keywords "radix astragali,fructus ligustri lucidi," "wolfberry fruit","limit","yam","seaweed","triangular","shows",""green husk" and "medicine”,"cinnamon","spreading hedyotis herb","act","these",:chicken’s gizzard-membrane,:leeches "," " whole worm".According to oralbioavailability(OB)≥30%,drug-likeoctant(DL)≥0.18 or druglikeoctant grade>0.49,the active ingredient database is selected according to the selection conditions of ADME(process of absorption,distribution,metabolism and excretion of foreign chemicals)of the active ingredient.The Candidate Target gene>0.8 was used as the Target protein screening condition to construct the Target protein database corresponding to the components.A database of disease-related genes was established using DisgeNET,GeneCards,and DrugBank databases,and the keyword "ovarian cancer" was used to retrieve ovarian cancer genes.(2)Network analysis:the components corresponding to the target protein and ovarian cancer related genes were matched and the intersection genes were selected to obtain the key genes.The key genes were introduced into string to obtain the protein interaction Network relationship,and the Network relationship was introduced into Cytoscape.The Network analysis was conducted using the Network Interpretation tool,and nodes of Degree>60 were screened as core genes.The "Bioconductor" and"clusterProfiler" packages in R language were used to conduct GO function enrichment and KEGG pathway enrichment of core genes.Through core gene mapping,the corresponding relationship among TCM,active components,core genes and pathways was established,and the network of "TCM-component-core target-pathway" was established.The Degree≥10 was used as the card value to screen the core Chinese medicine and core components in the network.2 Experimental study on the antitumor effect of YHJD on ID8 bearing mice transplanted with tumor(1)Build a mouse model of ovarian cancerFemale C57BL/6 mice were inoculated with 1x107 ID8 mouse ovarian cancer cells under the right upper arm,a total of 30 mice.After successful modeling,the mice were randomly divided into 5 groups:chemotherapy group(cisplatin),core Chinese medicine group(YHJD),Traditional Chinese medicine group(Cisplatin+YHJD),Chinese medicine group(YHJD),and simple tumor-bearing group(normal saline),with 6 mice in each group.(2)To observe the antitumor effect of YHJD on ID8 tumor-bearing miceThe general conditions of mice were observed daily,and the average daily intake,body weight and tumor length and diameter were measured once a week.Tumor inhibition experiments were completed when the tumor length diameter was>1cm in the tumor bearing group alone,tumor weight was weighed after sampling,and tumor inhibition rate of each treatment group was calculated[tumor inhibition rate=(average tumor weight of the tumor bearing group alone-average tumor weight of the treatment group)/average tumor weight of the tumor bearing group alone ×100%].3.mRNA microarray combined with network pharmacology for differential efficacy mechanism of YHJD in ovarian cancer(1)Data acquisition:300 patients with stage ⅢC-Ⅳ advanced ovarian cancer taking YHJD were included,and peripheral blood was extracted for RNA extraction and preservation after taking the drug for half a year.After follow-up,the 8 patients with the longest progress-free survival and the 7 patients with the shortest survival were selected for mRNA chip detection,and the gene with P<0.05 and differential multiple>1.2 was selected as the differentially expressed gene related to the efficacy of YHJD in ovarian cancer treatment.(2)The differentially expressed genes related to efficacy were intersected with the target proteins corresponding to the active components of YHJD obtained in the first study and the genes related to ovarian cancer disease to obtain the core genes with different curative effects.The core genes were imported into String to construct the protein interaction network,and the R language was used to obtain the Degree ranking of the core genes.Core genes were enriched using R language for GO function enrichment and KEGG pathway enrichment.By mapping the core genes related to different curative effects with Chinese medicine,active components and pathways,a network of "Chinese medicine-component-core target-pathway" related to different curative effects was constructed.RESULTSI.Network pharmacology study on the action mechanism of YHJD in the treatment of ovarian cancer(1)Data acquisition:YHJD screened out 193 active components conforming to ADME conditions from TCMSP,ETCM,BATMAN-TCM and TCMID databases.YHJD screened out 877 target proteins corresponding to the eligible active components from the TCMSP,ETCM and Batman-TCM databases.There are 18,483 genes related to ovarian cancer collected in DisGeNET,GeneCards and DrugBank database.(2)Network analysis:The active components of YHJD correspond to target proteins and genes related to ovarian cancer,and 676 key target genes of YHJD for the treatment of ovarian cancer were intersected.Important target genes were introduced into String and Cytoscape,and 123 tumour-related core genes such as IL6,VEGFA,TNF,MMP9,FN1,and angiogenesis were obtained with Degree>60 as the card value.On core genes GO,according to the function of enrichment YHJD treatment of ovarian cancer at the core of the genes involved in nuclear receptor function,activity of transcription factors,near RNA polymerase Ⅱ promoter biological processes.Enrichment of the KEGG pathway of the core gene showed that the core genes of YHJD for ovarian cancer treatment were involved in TNF signaling pathway,HIF-1 signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,IL-17 signaling pathway and other tumor-related pathways such as immunity and angiogenesis.To evaluate the Degree of Chinese medicine in the network of "Chinese materia Medica-components-core target-pathway",with the Degree≥10 as the calcine value,the core Chinese medicine of YHJD,fructus lyciI,scutellariae,artemisia annua,astragalus,Chinese yam,quercetin,kaempferol,baicalein,luteolin and other 10 active ingredients are the core active ingredients of YHJD in the treatment of ovarian cancer2 Experimental study on the antitumor effect of YHJD on ID8 bearing mice transplanted with tumor(1)A C57BL/6 mouse xenograft tumor model with ID8 ovarian cancer cell inoculations of 1×107 per mouse was constructed.All the models were successfully constructed one week after inoculation,and the drug administration was started and the growth status was observed.(2)After 11 weeks of administration of tumor bearing mice,the shortest tumor length diameter of each mouse in the tumor bearing group alone was 1cm,tumor inhibition experiment was stopped,and tumor inhibition rate was measured and calculated[tumor inhibition rate=(average tumor weight in the tumor bearing group alone-average tumor weight in the treatment group)/average tumor weight in the tumor bearing group alone ×100%],and the results showed that(2.1)The intake of mice in the core TCM group,the combined TCM group,the TCM group and the simple tumor-bearing group was higher than that in the chemotherapy group,with statistically significant differences[(2.86±0.49),(2.46±0.32)g,(2.76±0.38)g,(2.65±0.35)g vs(2.14±0.31)g,all P<0.05].The core Chinese medicine group was larger than the combined Chinese and Western medicine group,and the difference was statistically significant[(2.86±0.49)g vs(2.46±0.32)g,P<0.05].(2.2)The body weight of mice in the core TCM group,the combined TCM group,the TCM group,and the tumor-bearing group was higher than that of the chemotherapy group,with statistically significant differences[(22.49±0.85 g,(20.45±0.53)g,(21.48±0.86)g,(20.86±0.62)g vs(19.14±1.56)g,all P<0.05].The difference between the core TCM group and the combined Chinese and Western medicine group was statistically significant[(22.49±0.85)g vs(20.45±0.53)g,(20.86±0.62)g,all P<0.05](2.3)The tumor volume of mice in each group increased gradually over time.At the end of the experiment,the tumor volume of the chemotherapy group and the combined Chinese and Western medicine group was smaller than that of the simple tumor-carrying group,with statistically significant differences[(271±67)mm3,(21 8±128)mm3 vs(510±226)mm3,all P<0.05].(2.4)The tumor inhibition rate of each group was 32.01%in the core TCM group,43.14%in the chemotherapy group,51.09%in the TCM group,and 24.45%in the TCM group3.MRNA microarray combined with network pharmacology for differential efficacy mechanism of YHJD in ovarian cancer(1)Data acquisition:1157 genes related to differential efficacy of YHJD were obtained with P<0.05 and multiple>1.2 as the card value.(2)Network analysis:the 1157 differentially expressed genes were intersected with ovarian cancer related genes retrieved from the database and target proteins corresponding to YHJD in the second study,and 42 core genes related to the differentially expressed efficacy of YHJD in the treatment of ovarian cancer were obtained.Using R language tools in sorting and show Degree,display the AHR,TIMP1,NFE2L2,CCNB1,PARP1,MCL1,MDM2 gene in the core of such as are more important.The GO function of core genes is enriched in biological processes such as the activity of sodium ion transmembrane transporter,MAP kinase,nuclear receptor transcriptional coactivation,nucleotide enzyme activity,MAP kinase activity,hormone receptor binding and nuclear receptor binding.The enrichment of KEGG pathway of core gene showed that different therapeutic effects of ovarian cancer were mainly related to the following pathways:platinum resistance,p53 signaling pathway,the influence of cytochrome P450 on exogenous drug metabolism,cell apoptosis and other signaling pathways.A network of "TCM-component-core target-pathway" related to differential efficacy was constructed.CONCLUSIONS1.The core active components of YHJD in the treatment of ovarian cancer may be quercetin,kaempferol,baicalein,luteolin,etc.,while the core Traditional Chinese medicine may be astragalus membranaceus,Chinese yam,fructus lycii,scutellaria barbatae,and artemisia annua.2.YHJD may inhibit ovarian cancer by regulating IL6,VEGFA,TNF and other genes related to immunity and angiogenesis;AHR and other drug-resistant genes may be regulated genes in the dominant population.3.YHJD may inhibit ovarian cancer through regulating TNF signaling pathway,HIF-1 signaling pathway,MAPK signaling pathway,etc.,which are related to immunity and angiogenesis;Among them,the signaling pathways that regulate platinum resistance pathways and cytochrome P450,etc.and affect drug metabolism and efficacy may be the pathways regulated in the treatment of the dominant population.4.YHJD core Traditional Chinese medicine obtained from network pharmacology analysis has an inhibitory effect on tumor growth of tumor-bearing mice,and can enhance the anti-tumor effect of chemotherapy and improve the quality of life caused by tumor-bearing mice and chemotherapy.The results of network pharmacology analysis are reliable. |
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