Study On The Mechanism Of Inhibitory Effect Of The Effective Extraction Fractions Of Wenxia Formula On The Growth And Invasion Of Lung Cancer By Regulating CAFs-Mediated Hh-Gli1 Signaling Pathway

Objective:This study aims to investigate the pharmacodynamic material basis and anti-tumor mechanism of the effective anti-tumor extraction fractions from Wenxia Formula by building the model of nude mice into which tumor cells were subcutaneously transplanted and using analytical chemistry,network pharmacology,small animal imaging and molecular biology,to provide a scientific basis for its clinically applied..Methods:1.Solvent extraction was used to initially extract the ingredients of Wenxia Formula.The fractions were extracted with petroleum ether,dichloromethane,ethyl acetate and nbutyl alcohol,respectively.MTT assay was used to screen the anti-tumor activity of these fractions and decoction in vitro by lung cancer A549 cells,H1299 cells,H460 cells and H520 cells,to obtain the effective anti-tumor extraction fractions of Wenxia formula.2.Liquid chromatography-mass spectrometry(UHPLC-Q-Orbitrap HRMS)was used to collect the information of positive and negative ions in the test solution of the effective anti-tumor extraction fractions of Wenxia formula.And the chemical ingredients of the effective anti-tumor extraction fractions of Wenxia formula were analyzed qualitatively by the molecular formula,molecular weight,retention time and the rules of fragmentation in the mass spectrum of the compounds in the mzCloud.3.The network pharmacology and molecular docking were used to predict the mechanism of the effective anti-tumor extraction fractions of Wenxia formula.SEAware software was used to predict the potential target of these fractions;A compound-targetpathway network and PPI network were built by Cytoscape.Besides,GO analysis and KEGG analysis of core targets that were selected by searching David Database were performed,and molecular docking for core targets was performed by Molecular Operating Environment(MOE).4.Animal model of lung cancer in which A549 cells were implanted into nude mice was built by small animal imaging.These mice were divided into 7 groups:A549 group,model group,cis-platinum group,high-medium-low dose group(effective anti-tumor extraction fractions of Wenxia formula)and cyclopamine group.After 5 weeks of a drug intervention,the physical status,changes in weight of the animals were observed,and the tumor growth in nude mice was observed continuously by animal imaging.At the end of the experiment,the mice were killed,the tumors were removed,weighed and then stained with HE to observe the anti-tumor effect of effective extraction fractions of Wenxia formula.5.Based on the predictions by network pharmacology and molecular docking,the CAFs-mediated Hh-Glil signaling pathway was taken as an entry point in this study.The expression levels of ?-SMA,FAP,FN,ColIV,E-cadherin,N-cadherin,Vimentin,MMP2,MMP9 and TIMP1 in A549 xenografts were detected by immunohistochemistry,immunofluorescence,Western Blot and qRT-PCR.Besides,the expression levels of SHH,PTCH1,SMO and Gli1 in Hh-Gli1 signaling pathway were also detected to investigate the anti-tumor mechanism of the effective extraction fractions of Wenxia formula.Results:1.The results of in vitro anti-tumor activity screening test found that the fractions of petroleum ether,dichloromethane,ethyl acetate and n-butyl alcohol extracts inhibited the proliferation of A549 cells,H1299 cells,H460 cells and H520 cells to varying degrees.Besides,the fraction of ethyl acetate extract was proved to be more effective in inhibiting these four kinds of lung cancer cells in vitro than the other three extracts.The IC50 value to A549 cells was 198.7±1.25 ?g/mL,and there was a dose-effect relationship between the dose and the inhibitory rate.2.UHPLC-Q-Orbitrap HRMS method was adopted to identify 193 compounds of the fraction of ethyl acetate extract(mzCloud best match score>80),mainly including esters,phenols,ketones and alkaloids.Among them,the chemical ingredients of Angelica mainly include ferulic acid,senkyunolide H,chlorogenic acid and oleanolic acid.Rheum emodin,aloe-emodin,catechinic acid are the main chemical ingredients of rheum officinale.The key ingredient of Aconitum carmichaeli Debx is aconitine.The chemical ingredients of Panax ginseng mostly include kaempferol,isokaempferol and quercetin..3.The potential anti-tumor targets of CXCR4,LPAR1,SMO,CXCR2 and MMP2 were predicted by network pharmacology in the effective ingredients from the fraction of ethyl acetate extract.The possible signaling pathways mainly include the cancer pathway,PI3K-Akt pathway,neuroactive ligand-receptor interaction pathway and virus carcinogenesis pathway.The main biological processes are protein phosphorylation,protein self-phosphorylation,positive transcriptional regulation and DNA-templated chemistry,negative regulation of cell proliferation.The molecular functions mainly involve ATP binding,protein binding,protein kinase binding,protein-serine/threonine kinase activity and protein tyrosine kinase activity.The results of molecular docking showed that all of the first five core targets could dock with many ligand compounds.It can be seen from the docking scores and binding free energy that LPAR1,SMO and MMP2 had stronger binding with ligand compounds.4.The fraction of ethyl acetate extract could improve the physiological status and pathological changes of the transplanted tumor in nude mice,but had no obvious effect on the body weight,spleen and biochemical index of the tumor-bearing nude mice.It inhibited the growth and invasion of A549 xenografts to varying degrees(P<0.05,P<0.01),and showed dose-dependent.5.The fraction of ethyl acetate extract could reduce the expression level of ?-SMA,FAP,FN,Co1?,N-cadherin,Vimentin,MMP2,MMP9 and mRNA in A549 xenografts(P<0.01,P<0.05),increase the expression level of E-cadherin,TIMP1 and mRNA(P<0.01,P<0.05),and decrease the expression level of PTCH1,SMO and Gli1 and mRNA in Hh-Gli1 signaling pathway(P<0.01,P<0.05).?Conclusion:The fraction of ethyl acetate extract could inhibit the growth and invasion of A549 xenografts to some extent.The mechanism may be related to its regulation of the CAFsmediated Hh-Gli1 signaling pathway to inhibit epithelial-to-mesenchymal transition(EMT),damage to the extracellular matrix(ECM)and remodeling.