| Objectives:The aim of my study was to study the anti-migration and anti-invasion effects and the possible mechanism of Oldenlandia diffusan Willd.(OD),Sculellaria barbata(SB)and their herb pairs on human glioblastoma cells,and to screen the best antitumor extract solvent and the proportion of herb pairs,and to compare the difference of action between single drugs and herb pair.Methods:1.OD,SB and their herb pairs extracts were prepared with different proportion(OD:SB 1:1,1:2 and 2:1)by reflux extraction using three solvents:water,40%ethanol,80%ethanol.Then the inhibition effects of herb extracts on of glioma cells proliferation were explored by MTT method.The extracts were analyzed by UPLC(Ultra Performance Liquid Chromatograph).UPLC detection protocol was established by examining the detection wavelength,mobile phase and acid.Fifteen extractions were analyzed by UPLC,and the prints were compared.Finally,the optimal extraction solvent and the best herb pair proportion were screened by the results of MTT assay and UPLC analysis.2.The anti-migration and anti-invation effects of OD,SB and their herb pairs on human glioma cells U251 and U87 were investigated.The MTT assay,live/dead staining and FCM(flow cytometry)assay were performed to study the effects of OD,SB and the herb pairs with concentrations of 0,0.5,1,2,4 and 8 mg/mL on cell proliferation and apoptosis of U251 and U87 cells.Afterwards,the drugs at lower concentrations(0.5,1 and 2 mg/mL),with no obvious influence on cell proliferation,were chosen to be used in the migration and invasion study.Scratch wound healing assay and Transwell assays were used to investigate the effects on cells migration and invasion under two dimentional culture.The invasion assay of cells cultured in three dimentional was conducted on a microflodic chip with multicellular spheroids.3.UPLC-Q-TOF/MS was employed to explore and describe OD and SB extracts.4.A systematic network pharmacological analysis was used to analyze the possible anti-glioma active constituents,targets and mechanism of OD and SB.According to the pharmacokinetic parameters of each component provided by the platform,the compounds that appear to have OB(Oral bioavailability)30%and DL(Drug similarity)0.18,respectively,are selected as the effective components.On the TCMSP platform,the targets of the active components of OD and SB were further searched,and the network diagram of the active components and their targets was constructed by Cytoscape software.A disease-target network graph was constructed on Cytoscape software,using tools such as OMIM,TTD and GEO to find therapeutic targets related to glioma.On this basis,Cytoscape Bisogenet or STRING database was used to analyze the interaction relationship between drug target and disease target protein,and construct PPI(protein interaction)graph was generated.Using DAVID or STRING database,GO annotation analysis could be conducted,and KEGG pathway analysis could screen out the relevant pathways of anti-glioma cell migration and invasion of OD and SB,which accounted for the mechanism of the drug effects..Results:1.The results of MTT showed that 80%ethanol extracts exhibited the best inhibition effects on glioma cell proliferation in three solvent extracts,and the 1:2 herb pair displayed the best drug effects in three herb pair proportions.According to the results of UPLC analysis,there were no obvious new substances peaks in herbal pairs prints compared with single herbs.However,the area and peak height of common peaks were different and 1:2 herb pair was the greatest in three proportions.This would account for the better effect of 1:2 pair than other pairs in MTT assay.Therefore,we studied the migration and invasion with 80%ethanol extracts of OD,SB and their 1:2 herb pair.2.OD,SB and 1:2 herb pair extracts exhibited obvious anti-proliferation and pro-apoptosis effects on U251 and U87 cells based on the results of MTT assay,live/dead staining and FCM apoptosis assay,in concentrations-related manner.The results showed that the lower concentrations(0.5,1 and 2 mg/mL)extracts had no obvious influence on cell proliferation,therefore used in later experiments.In the two-dimentional cell migration and invasion tests,OD,SB and 1:2 herb pair showed obvious inhibition effects with concentrations of 0.5,1,2 mg/mL on glioma cells,and similar results were found on the cells cultured on three-dimensional perfused microfluidic chip.3.OD and SB were analyzed by the UPLC-Q-TOF-MS detection.Under positive ion mode,35 and 42 compounds were detected from the extracts of OD and SB,respectively.4.The network pharmacology analysis was adopted to explore the effective ingredients,action targets and related mechanisms of the anti-human glioma migration and invasion of OD and SB.Twenty-four and 29 active components of OD and SB were screened out,and the corresponding target of each active component was collected,and the network diagram of component-target was constructed by Cytoscape software.By searching from multiple disease target databases,72 targets of human glioma were sorted out,and constructed PPI network diagrams of the interaction relationship between targets of OD and SB components-human glioma disease target proteins,respectively.GO annotation and KEGG pathway analysis were carried out on node proteins in PPI network diagram,and the results showed that OD may inhibit the migration and invasion of glioma cells by participating in the regulation of MAPK pathway,Wnt signaling pathway and microtubule information skeleton.This anti-glioma proliferation and pro-apoptosis effects may related to its involvement in the regulation of cell cycle arrest and negative regulation of cell proliferation,the intervention of endogenous apoptosis pathways and pro-apoptosis process,and the classical P53 regulation.The anti-human glioma cell migration and invasion mechanism of SB may be related to the regulation of the adhesion spot,VEGF,MAPK pathway.In addition,it is involved in PI3K-AKT,JAK-STAT,p53,Ras and other signaling pathways as well as cell cycle regulation.All these biological behaviors are related to its anti-glioma cell proliferation and pro-apoptosis effects.Conclusions:OD,SB and their herb pairs could siginicantly restrain migration and invasion of human glioma cell.Since no new peaks appeared in UPLC analysis,we considered that the drug took effects as the results of overlay effects of two single herbs.Network pharmacological studies showed that OD may inhibit the migration and invasion of glioma cells by participating in the regulation of MAPK pathway,Wnt signaling pathway and microtubule information skeleton.Moreover,the anti-human glioma cell migration and invasion effect of SB may be related to its regulation of adhesion spot,VEGF,MAPK signal path.The results suggested that the effects of OD,SB and their herb pairs may provide new drug clues for the treatment of migration,invasion and recurrence of human malignant glioma. |
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