| ObjectiveTriple-negative breast cancer is the most aggressive subtype of breast cancer,and its special molecular background determines that it cannot benefit from traditional hormone therapy or targeted therapy.Successful clinical management remains an elusive topic,and new clinical treatment strategies are urgently needed to improve the situation.The traditional Chinese medicine compound Loulu Powder has a good clinical efficacy basis,but it lacks the effective elucidation of the modern mechanism.Based on the topic of the intervention of Loulu Powder in triple-negative breast cancer,the concept of integrated pharmacology model was introduced.Starting from the prediction module and the experimental verification module,the pharmacodynamic material basis of Loulu Powder and the risk target of triple-negative breast cancer were analyzed.Meanehile,the potential biological mechanism of Loulu Powder intervening in triple-negative breast cancer was also explored.Method1.The triple-negative breast cancer patients who were treated in our hospital were retrospectively screened.The visit data and medical record information were extracted,and the syndrome distribution characteristics of triple-negative breast cancer patients were analyzed.Further,the chemical constituents of Loulu Powder were identified based on Q-Orbitrap high-resolution LC/MS,and the targets were predicted based on the relevant "traditional Chinese medicine-component-target" database,and a molecular-target network was constructed.The mechanism of effectiveness of Loulu Powder was explored and analyzed from the molecular level.2.The sequencing data of triple-negative breast cancer was obtained from high-throughput databases,the big data was analyzed and processed based on bioinformatics algorithms,and the key risk target information of triple-negative breast cancer was obtained by dimensionality reduction screening.Data set validation was performed for key targets and pathways,and molecular docking scoring with key components of Loulu Powder was carried out to screen drug targets with effective docking interaction patterns.We further collected the pathological samples of triple-negative breast cancer patients in the hospital to evaluate the key target on protein level,and finally determined the key risk targets and pathways of Loulu Powder intervening in triple-negative breast cancer.3.In vitro and in vivo basic experiments were used to analyze and verify the potential molecular mechanism of Loulu Powder’s intervention in triple-negative breast cancer.To detect the effect of Loulu Powder on the proliferation,migration and invasion of triple-negative breast cancer cell lines;to detect the intervention of Loulu Powder on triple-negative breast cancer cell cycle and apoptosis based on flow cytometry;to construct mouse model and explore the inhibitory effect of Loulu Powder on tumor growth of triple-negative breast cancer mice,as well as its impact on the survival cycle,survival status,etc.;finally,western blot was uesd to explore the intervention effect of Loulu Powder on key targets.Results1.Patients with triple-negative breast cancer who came to our hospital for treatment and were admitted for treatment from October 2017 to December 2021 were collected.After screening according to the inclusion and exclusion criteria,a total of44 triple-negative breast cancer patients were retrospectively analyzed.The results showed that there were 6 patients with Chongren-shitiao syndrome,24 patients with Ganyu-tanning syndrome,and 14 patients with Zhengxu-duchi syndrome.This outcome is also consistent with previous studies,that is,the syndrome types of most triple-negative breast cancer patients are concentrated in the syndrome of Ganyu-tanning and Zhengxu-duchi,especially in patients over 50 years old.Furthermore,based on Q-Orbitrap LC/MS analysis and the database exploration of active ingredients of traditional Chinese medicine,51 key components of Loulu Powder were excavated,and 392 targets were analyzed.The enriched pathways were concentrated in the pathways related to tumorigenesis and development.2.According to the inclusion criteria of immunohistochemistry and fluorescence in situ hybridization,156 triple-negative breast cancer patients were screened from the TCGA database cohort,and 10,719 differentially expressed genes were obtained after differential analysis screening and standard truncation.WGCNA was further performed for dimensionality reduction screening and obtained a significant risk association module(r=0.84,P=1e-72).Related genes were further analyzed for enriched pathways,and the PI3K/AKT signaling pathway was found to be involved in the regulation of triple-negative breast cancer by Loulu Powder.Molecular docking discovered a good docking mode between EIF4EBP1 and BCL2A1 and the effective components of Loulu Powder.The risk performance of the two targets in triple-negative breast cancer was further analyzed based on the immunohistochemistry of clinical samples from triple-negative breast cancer patients,and the results showed high expression in triple-negative breast cancer compared with normal tissues,which was consistent with the transcriptome level.This suggested that EIF4EBP1 and BCL2A1 are associated with risk of triple-negative breast cancer.3.The in vitro cell experiments were carried out based on the classic triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468.The MTT experiment clarified the significant activity inhibition of Loulu Powder on triple-negative breast cancer cells.The IC50 s at 24 hours were 4.919mg/ml and5.156mg/ml,respectively;In addition,the two dose groups of 2 and 4 mg/ml Loulu Powder significantly inhibited the migration and invasion abilities of MDA-MB-231 and MDA-MB-468 cell lines(P<0.05);The cell line MDA-MB-468 showed a significant apoptosis outcome when 2 mg/ml of Loulu Powder was intervened,and this situation was more significant in the MDA-MB-468 cell line,as the drug concentration of 1 mg/ml showed a significant apoptosis outcome(P<0.01);For cell cycle intervention,in MDA-MB-468 cells,the trend appeared after the intervention at4 mg/ml and 8 mg/ml,with a statistically significant decrease in the proportion of cells in the G1 phase and an increase in the proportion of cells in the G2/M phase(P<0.01).In MDA-MB-231 cells,the increase trend of cells in G2/M phase was more sensitive than that in MDA-MB-468 cells,and the increase of cells in G2/M phase began to appear from the concentration of 1 mg/ml(P<0.05).In vivo experiments in tumor-bearing mice found that after the intervention of Loulu Powder,the low-dose group has already showed an effective tumor inhibition effect,and the tumor volume and weight were significantly reduced compared with the blank control group,and the difference was statistically significant(P<0.05).This inhibitory effect became more pronounced with increasing drug concentration(P<0.01),and the high-dose group showed similar outcomes to the positive control group.Survival curves were drawn to visualize the survival of tumor-bearing mice,and the results showed that the mice in the blank control group had the shortest survival period and higher mortality,while the mice in the middle-dose,high-dose and positive control groups survived relatively well,and the survival situation was not much different,which suggested that Loulu Powder has a good effect on tumor intervention in vivo and protective effect on the body;Western blot results showed that the intervention of Loulu Powder significantly inhibited the expression of PI3K/AKT pathway,and the downstream key factor BCL2A1 and 4EBP1 expression were also significantly reduced.Conclusion1.In this study,the distribution characteristics of syndrome types in patients with triple-negative breast cancer were analyzed,which provided the theoretical basis of traditional Chinese medicine for the effectiveness of the Loulu Powder.The clinical application of Loulu Powder in triple-negative breast cancer patients with typical characteristics of Ganyu-tanning and Zhengxu-duchi syndrome was appropriate.2.Based on the effective exploration of Q-Orbitrap LC-MS and traditional medicine target database,it is suggested that the key components and targets of Loulu Powder are effectively enriched in the pathways related to the occurrence and development of tumors,which provided a basis for the study of the intervention mechanism of Loulu Powder.3.High-throughput data analysis of triple-negative breast cancer and algorithmic dimensionality reduction quality control screening identified the potential roles of PI3K/AKT signaling pathway and key hubs EIF4EBP1 and BCL2A1 in the intervention of Loulu Powder in triple-negative breast cancer.Associated risk associations were validated at both transcriptome and protein levels.4.In vitro and in vivo experiments showed that Loulu Powder could interfere with the progression of triple-negative breast cancer by regulating the PI3K/AKT signaling pathway.5.The integrated pharmacology model with network pharmacology combined with bioinformatics as the pre-prediction module and basic experiment as the verification module is considered to be meaningful,and can be released into effective discoveries of traditional Chinese medicine intervention mechanisms on other disease. |
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