Network Pharmacology And Transcriptome Study On The Molecular Mechanisms Of Huan Qi-Ezhu Formula For Lung Cancer Treatment

Purpose:To further clarify the mechanism of Huang Qi-Ezhu formula inhibiting the growth and metastasis of lung cancer,we used the methods of network pharmacology and transcriptomics to predict the target and signaling pathway from the perspective of systems biology,and explored the mechanism of Huang Qi-Ezhu formula affecting tumor immunity and blood vessels in vivo intervention experiments.Methods:1.Network pharmacological research:High performance liquid chromatography results and the Traditional Chinese Medicine Systems Pharmacology（TCMSP）database were used to build database.The ADME attribute values of OB≥30%and DL≥0.18 were used to screen the active ingredients.Targets were obtained using the Swiss Target Prediction and TCMSP databases.Proteins related to LC were retrieved from the Gene Cards database.A protein-protein interaction（PPI）network was constructed in the STRING database,and the top 10 core targets were obtained.The David database was used for GO function analysis,and KEGG pathway enrichment analysis.2.Transcriptome studies:Subcutaneous xenograft tumor mice model were conducted using LLC,and were randomly divided into model group and Huang Qi-Ezhu formula group.The animals were given normal saline（NS）or the respective Chinese medicine decoction liquid by gavage once every morning for 14 days.Total RNA was extracted from tumor tissue with TRIzol kit and sequenced by Illumina.The results were screened according to the difference significance criteria（the difference gene expression changed more than 1.5 times and qvalue（FDR,Padj）<=0.05）.Perform Clustering analysis,GO enrichment analysis and KEGG signaling pathway enrichment analysis of differentially expressed genes.The differential genes of network pharmacology and transcriptome were analyzed by STRING database for PPI interaction,and key signaling pathways were found and analyzed.3.In vivo intervention experiment:Subcutaneous xenograft tumor mice model were conducted using LLC,and were randomly divided into model group,Positive drug group and Huang Qi-Ezhu formula group（3:1）.The animals were given normal saline（NS）or the respective Chinese medicine decoction liquid by gavage once every morning for 14 days.Positive drug group were intraperitoneally injected with Cisplatin prepared with normal saline at 5mg/kg on days 5,7,and 9 after the subcutaneous injection of LLC tumor cells.On the 15th day,Tumor vessels were examined by contrast-enhanced ultrasound using one-time mass injection of microvesicles.Tumor weight index,tumor inhibition rate,thymus index and spleen index were Weighed and calculated.Tumor histopathology and Apoptosis of tumor cells were detected by H-E staining and TUNEL assay.The coverage rate of tumor vascular pericytes was detected by Fluorescent Antibody Assay.Proportion of CD₄⁺T and CD₈⁺T in spleen,TH1 and TH2 type cytokines in tumor tissues were analyzed by flow cytometry and ELISA.Immune and vascular related genes expression were validated and analyzed by RT-q PCR.Results:1.Network pharmacological study:Building a drug interaction network-composition-targets-disease by using the method of network pharmacology,we found 33 kinds of effective active ingredients of Huang Qi-Ezhu formula,and showed that 172 target proteins and multiple pathways play an important role in the treatment of lung cancer.AKT1,VEGFA,IL6 other 7targets were at the core of the network,plays multiple important roles in gene transcription regulation,involving a variety of cell components and molecular functions,and is closely related to cell proliferation,regulation and other biological processes.In addition PI3K/AKT,HIF1 and TCR signal pathways were the most important pathway.2.Transcriptomics studies:With the technique of transcriptome,we found that there were241 different genes involved in the regulation of cell adhesion,proliferation,migration,angiogenesis and immunity in the Huang Qi-Ezhu formula compared with the model group.Go enrichment analysis showed that in the biological process of Huang Qi-Ezhu formula inhibiting lung cancer related to T cell activation,and KEGG enrichment results also showed that the most significant signal pathway related to immunity.3.In Huang Qi-Ezhu formula group,the microvessel density decreased,the pericyte coverage increased,and the blood perfusion of tumor vessels improved,indicating that it promoted the normalization of tumor vessels.In the aspect of immunity,the transcription levels of CCL5,CXCR3,CD4 and CD8a genes in tumor tissues were increased in Huang Qi-Ezhu formula group,while the number of suppressive immune cells in spleen of peripheral immune organs was decreased and the number of CD8+T cells was increased.Conclusion:Based on the analysis of previous studies and the results of network pharmacology and transcriptomics study,it is suggested that the anticancer effect of Huang Qi-Ezhu formula is closely related to its role in regulating tumor immunity and influencing tumor blood vessels.Vivo experiments verified the conjecture,indicating that the mechanism of the compatibility of anti-cancer is to affect the tumor microenvironment by regulating the tumor blood vessels and immune function at the same time,so as to achieve the effect of inhibiting tumor growth and metastasis.Our work provided the key research direction to further clarify the anti-cancer mechanism of Huang Qi-Ezhu formula.