| Objective:Cervical cancer is one of the most common malignant tumors in women,with high incidence and postoperative recurrence rate,which is the main cause of female cancer death.In recent years,the incidence of cervical cancer tends to be younger,which seriously threatens the lives and health of women.Supplementing qi and activating blood is the basic principle of traditional Chinese medicine in the treatment of cervical cancer.Our previous study found that Bushen Yiqi Huoxue Formula（Formula 1）composed of Epimedii Folium,Astragali Radix,Rehmanniae Radix,Angelicae Sinensis Radix and Callerya reticulata（Benth.）Schot.has the ability to correct the leukocyte injury after chemotherapy and promote the recovery of immune function.In addition,the pre-experiment proved that Formula 1（F1）could significantly inhibit the proliferation activity of cervical cancer HeLa cells.Salviae Miltiorrhizae Radix et Rhizoma is a commonly used traditional Chinese medicine with the effect of supplementing qi and activating blood,and modern pharmacological studies have found that Salviae Miltiorrhizae Radix et Rhizoma and its extracts have significant inhibitory effect on cervical cancer.Therefore,on the basis of F1,Salviae Miltiorrhizae Radix et Rhizoma was added to form Modified Bushen Yiqi Huoxue Formula（Formula 2）to enhance the effect of F1 on supplementing qi and activating blood.Therefore,this thesis aims to systematically clarify the inhibitory effect and mechanism of F1 and F2 on cervical cancer metastasis,reveal the synergistic effect of Salviae Miltiorrhizae Radix et Rhizoma,and provide a theoretical basis for the clinical application of Yiqi Huoxue Formula in cervical cancer.Contents and Methods:The network pharmacology method was used to construct the drugdisease molecular target networks of the main active components of F1 and F2 for inhibiting cervical cancer.Liquid chromatography-mass spectrometry was used to elucidate the active components of traditional Chinese medicine contained in the extracts of F1 and F2.The effects of F1 and F2 on the proliferation,cell number,colony formation ability,F-actin cytoskeleton structure,cell migration and invasion ability of cervical cancer HeLa cells were detected by CCK-8 assay,cell counting,colony formation assay,immunofluorescence staining,wound healing mobility assay and Transwell cassay.Annexin V-FITC/PI assay was used to observe the effect of F2 on the apoptosis of cervical cancer HeLa cells.Flow cytometry and laser confocal microscopy were conducted to detect the effect of F2 on autophagy markers LC3B,autophagy flow and co-localization of mitochondria and lysosomes in cervical cancer HeLa cells.RNA-seq assay and Western Blot were used to detect the effect of F1 and F2 on the transcription and protein expression of cervical cancer HeLa cells.Finally,the effect of F1 and F2 on the metabolic spectrum of cervical cancer HeLa cells was detected by 1HNMR metabolomics assay.Results:With OB≥30%and DL≥0.18 as screening conditions,F1 contained 60 active ingredients and F2 contained 123 active ingredients.The common active ingredients of F1 and F2 were Calycosin,Quercetin,Kaempferol,Icariin and β-sitosterol,etc.F2 also contained active components of Salviae Miltiorrhizae Radix et Rhizoma,such as Cryptotanshinone,Isotanshinone Ⅱ and Tanshinone ⅡA.These active ingredients mainly achieved the anticervical cancer effect by regulating targets such as IL6,TNF,IL1B,CCL2 and CSF2,and then targeting biological processes such as negative regulation of apoptotic process,positive regulation of gene expression and positive regulation of cell proliferation,and signaling pathways such as pathways in cancer,apoptosis,PI3K-AKT signaling pathway and proteoglycans in cancer.Compared with F1,F2 was more concentrated in regulating apoptosis and PI3K-AKT signaling pathway.The results of liquid chromatography-mass spectrometry showed that F1 and F2 contained Ligustilide,Icariin and β-Sitosterol-β-D-glucoside and other active ingredients.F2 also contained cryptotanshinone,an active ingredient of Salviae Miltiorrhizae Radix et Rhizoma.The results in vitro showed that compared with the solvent control group,F1 and F2 significantly inhibited the proliferation activity and colony formation ability of cervical cancer HeLa cells in a concentration-dependent manner,and the effect of F2 was better than that of F1.Both F1 and F2（400 μg/mL and 800 μg/mL）could also regulate the cytoskeleton structure of F-actin,and then inhibit the migration and invasion of cervical cancer HeLa cells.Furthermore,F2（400 μg/mL and 800 μg/mL）significantly induced the apoptosis of cervical cancer HeLa cells and promoted the expression of autophagy marker LC3B,induced autophagy flow and increased the number of co-localization of mitochondria and lysosomes to activate mitophagy.The results of RNA-seq assay showed that F1 and F2 at 800μg/mL significantly affected the gene expression of cervical cancer HeLa cells,upregulated genes including autophagy-related genes:ATG5,ATG4A,ATG4C,and tumor glucose metabolism-related genes:PDHX,PDP1,etc.,down-regulated tumor glucose metabolism-related genes:HKI,HKII,PFKP,PFKL,etc.GO enrichment and KEGG pathway analysis showed that F1 and F2 were involved in the regulation of biological processes such as oxidoreductase activity,mitochondrial protein complex and cell-substrate adherens junction,and signaling pathways such as autophagy-animals,mitophagy-animals and oxidative phosphorylation.In addition,F1 and F2 regulated a total of 1174 differential genes in cervical cancer HeLa cells,which were mainly enriched in biological processes such as oxidoreductase activity,cell-substrate adherens junction,oxidative phosphorylation and mitochondrial ATP synthesis,and regulated signaling pathways such as proteoglycans in cancer,PI3K-AKT signaling pathway and oxidative phosphorylation in cancer.Western Blot results showed that F2 significantly promoted the expression of autophagy-related proteins Beclin-1,Atg5,Atg12 and Atg16L1,and increased the ratio of LC3B/A;after the addition of autophagy inhibitor chloroquine,the expression of Beclin-1,Atg5,Atg12 and Atg16L1 decreased,while the ratio of LC3B/A further increased.F2 also down-regulated the expression of glycolysis-related proteins HKI,HKII,PFKP,PKM2 and LDHA,and promoted the expression of PDH protein.Metabolomics results revealed that 800 μg/mL of F1 and F2 significantly affected 22 metabolites of cervical cancer HeLa cells,such as creatine,leucine,valine and taurine,etc.And these differential metabolites were mainly involved in glyoxylate and dicarboxylate metabolism,citrate cycle（TCA cycle）,pyruvate metabolism,glycolysis/gluconeogenesis and other biological processes.Conclusion:（1）F1 and F2 contained active ingredients of traditional Chinese medicine such as Ligustilide,Icariin and β-sitosterol,and may act on targets such as IL6、TNF、IL1B、CCL2 and CSF2,and then target biological processes such as negative regulation of apoptotic process,positive regulation of gene expression and positive regulation of cell proliferation,and signaling pathways such as pathways in cancer,apoptosis,PI3K-AKT signaling pathway and proteoglycans in cancer,so as to play an anti cervical cancer role.（2）F1 and F2 significantly inhibited the proliferation of cervical cancer HeLa cells,changed the cytoskeleton structure of cells and weakened the migration and invasion of cells in a concentration-dependent manner.F2 induced the apoptosis of cervical cancer HeLa cells,induced autophagy and increased the number of co-localization of mitochondria and lysosomes to activate mitophagy.（3）F1 and F2 significantly affected the gene expression of cervical cancer HeLa cells,including autophagy and glycolysis related genes.（4）F2 regulated the expression of autophagy and glycolysis-metabolism-related proteins in cervical cancer HeLa cells to induce autophagy and inhibit glycolysis-metabolism.（5）F1 and F2 significantly affected the metabolic spectrum of cervical cancer HeLa cells,such as glyoxylate and dicarboxylate metabolism,pyruvate metabolism,glycolysis/gluconeogenesis and so on. |
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