| Ligustrum Lucidum Ait.belongs to Ligustrum of oleaceae,which is widely distributed all over the world and is an important wild plant resource.Fructus Ligustri Lucidi is the fruit of Ligustrum Lucidum Ait..Fructus Ligustri Lucidi had high medicinal value and had the effects of nourishing liver and kidney and delaying aging.According to the domestic and foreign literature,it is found that Fructus Ligustri Lucidi contains bioactive substances,such as ursolic acid,oleanolic acid and ligustrin,which can resist inflammation and sterilization,reduce blood lipid,reduce blood glucose,regulate the immune function of the body,and have anti-cancer effects.The existing clinical and experimental results have found that Fructus Ligustri Lucidi has good antitumor effect.However,most of the current studies are aimed at the anticancer effect of ursolic acid,one of its main components.The chemical components of Fructus Ligustri Lucidi that exert antitumor activity have not been comprehensively analyzed,and the anti-cancer mechanism of other components has not been systematically reported.Based on this,this paper carries out relevant research from the following aspects,and has achieved phased results.(1)Analysis of bioactive substances from Fructus Ligustri Lucidi against breast cancer based on network pharmacology:Aiming at the breast cancer process of Fructus Ligustri Lucidi,through TCMSP,UniProt and GeneCards databases,and based on similarity algorithm,we screened effective bioactive components and predicted potential therapeutic targets.The tool of DAVID was used to analyze the function enrichment of targets,string database and Cytoscape software were used to analyze the protein-protein interaction between targets,and PPI network diagram was drawn.The results showed that 32 key targets of breast cancer were mainly from 8 bioactive components,ursolic acid and oleanolic acid.The target of oleanolic acid contains the common breast cancer treatment molecule ESR1(estrogen receptor 1),AR(androgen receptor),PGR(progesterone receptor),PPARG(peroxisome proliferator activated receptor gamma)and PRLR(prolactin receptor).The results of functional enrichment analysis showed that 32 key targets were mainly enriched in 9 significant pathways,37 biological processes,7 molecular functions and 4 cytological component functional categories.The results of target PPI network analysis showed that there were interactions among 15 target proteins in oleanolic acid,and the top five protein-protein interactions were CYP17A1-SRD5A1,AKR1C1-SRD5A1,ESR1-PGR,AR-NR3C2 and AR-NR3C1.(2)Extraction and purification of oleanolic acid from Fructus Ligustri Lucidi:In order to screen effective bioactive components of Fructus Ligustri Lucidi against breast cancer in early stage,oleanolic acid was selected for further study.using ultrasonic assisted extraction method and Fructus Ligustri Lucidi as raw material,the extraction process of oleanolic acid active components was optimized.Taking the yield of oleanolic acid as the response value,Box-Behnken central combination design principle and response surface analysis method were used to investigate the concentration of extractant,solid-liquid ratio,ultrasonic time The influence of ultrasonic power and other factors on the yield of oleanolic acid,so as to obtain the optimal extraction conditions of oleanolic acid through experiments;Oleanolic acid extract was purified by macroporous adsorption resin purification method,so as to obtain high-purity oleanolic acid for subsequent biological experiments.(3)Transcriptome analysis of oleanolic acid inhibiting breast cancer cells based on high throughput sequencing:the effect of oleanolic acid(OA)on breast cancer cell lines was studied at cellular level by using purified oleanolic acid obtained from Fructus Ligustri Lucidi.RNA-seq technique was used to explore the molecular mechanism of oleanolic acid inhibiting MCF-7 cells at the level of functional genomics.The results showed that 40 μM OA treatment could significantly inhibit the proliferation and induce the apoptosis of MCF-7 cells.Through analysis of RNA sequencing data quality and differentially expressed genes(DEGs),67 significantly downregulated genes and 260 significantly upregulated genes were identified to be involved in OA treatment of MCF-7 cells.Among these genes,43 unique DEGs were enriched in several signaling pathways and Gene Ontology terms,such as p53 signaling pathway,TNF signaling pathway and mTOR signaling pathway.Six downregulated genes,including THBS1,EDN1,CACNG4,CCN2,AXIN2 and BMP4,as well as six upregulated genes,including ATF4,SERPINE1,SESN2,PPARGC1A,EGR1 and JAG1,were selected as target genes in response to OA treatment.(4)Mechanism of oleanolic acid inhibiting breast cancer in mice:4T1-luc cells were subcutaneously injected into the chest wall of five week old female BALB/c mice to construct a tumor formation model of breast cancer.In order to detect the anti-tumor effect of OA,the mice were divided into OA group and model group,respectively,by continuously intragastric administration of OA(30mg/kg)and equal dose of corn oil.The weight of mice was weighed and recorded every day,and the tumor size was measured with vernier caliper;After 19 days of gavage,in vivo bioluminescence imaging of the breast tumor in mice was performed by Tanon-5200Multi multi-function imager.Then the mice were killed by spinal dislocations,and the tumors and their visceral tissues were removed from the mice and wei ghed and photographed.QPCR was used to detect the expression of 12 target genes screened by cell transcriptomics in OA group and model group.The morphological observation of mice showed that the body weight and tumor volume of OA treated mice decreased significantly;In vivo imaging of mice showed that the tumor fluorescence signal of OA treated mice decreased significantly;The tumor body observation results showed that the tumor surface of OA treated mice was smooth,the tumor size and weight were significantly reduced,the color of liver and spleen was darker and the weight decreased significantly.The above results show that OA treatment can significantly inhibit the development of breast tumors at the mouse level,but it also has some toxic and side effects;The expression patterns of six upregulated genes and six down regulated genes screened by RNA-seq and bioinformatics analysis were verified by qPCR test in mice,and revealed the targeting mechanism of OA in the treatment of breast cancer.In conclusion,in this paper,network pharmacology was first used to discover 8 bioactive components of Fructus Ligustri Lucidi against breast cancer,which involved 32 key targets,the top two were ursolic acid and oleanolic acid.High purity oleanolic acid was obtained from wild Fructus Ligustri Lucidi by ultrasonic assisted extraction and macroporous adsorption resin purification.The effect of oleanolic acid on breast cancer was revealed in vitro and in vivo.The key factors and pathways of oleanolic acid inhibiting proliferation and metastasis of breast cancer MCF-7 cells were further screened and identified by RNA-seq and molecular biology methods,and the mechanism of oleanolic acid inhibiting the development of breast tumor in mice was explained.All of these will provide certain theoretical basis and data support for the prevention and treatment of breast cancer by Chinese medicine.Based on these,vigorously developing the planting and plant resource protection of Ligustrum Lucidum Ait.in China and guiding the development and utilization of wild plant resources will help to give full play to the medicinal value of the anti-tumor effect of Fructus Ligustri Lucidi. |
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