| Background:Over the past 30 years,the team has been deeply engaged in the research of traditional Chinese medicine for the prevention and treatment of lung cancer,and has successfully developed the Yifei Sanjie pill(YFSJP).A series of clinical studies have confirmed that YFSJP can promote efficacy and attenuated side effects of routine treaments in patients with non-small cell lung cancer(NSCLC).And the pharmacological mechanism has also been explored in previous basic researches.However,the pharmacodynamic material basis remains unclear.Objective:To clarify the pharmacodynamic material basis of YFSJP against NSCLC and explore the mechanism of YFSJP against NSCLC metastasis,so as to provide proof for the clinical application and new drug development of YFSJP.Methods:1.Chemical analysis of YFSJP extractUltra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS)was applied for chemical analysis of YFSJP extract.PeakView Software and literature were used to analyze the mass spectrometry data and the chemical composition of YFSJP extract was finally obtained.2.Establishment of HPLC fingerprint of YFSJP extract and content determination of the four componentsHigh performance liquid chromatography-diode array detector(HPLC-DAD)technology was used to establish the fingerprint of YFSJP extract.The chromatographic condition optimization,methodology investigation(specificity,precision,stability,repeatability)were carried out respectively.The"Traditional Chinese Medicine fingerprint similarity evaluation system(2012 version)" was used to generate fingerprint and compare the similarity of different batches of chromatograms.The chemical composition was identified by comparing the UV absorption between the standards and the target peak.Then,the linear relationship and methodology(specificity,precision,stability,repeatability,recovery)were established.And the content determination of the four components in 10 batches of YFSJP extract were measured.3.Establishment of pretreatment method for cell dissociation solution samplesSolid phase extraction(SPE)was used to pre-treat the analytical samples.The effects of two SPE extraction columns and two elution methods on the recovery efficiency were compared,and the SPE treatment parameters with the best recovery rate were adopted.4.Establishment of A549-cell membrance chromatography(CMC)and identification of binding componentsFirstly,the toxicity of YFSJP extract on A549 cells was detected by counting kit-8(CCK8),and the relatively non-toxic concentration was selected as the following concentration for CMC.The number of washes in CMC was optimized,and samples were analyzed after SPE process.The last washing solution was the one without chromatographic peaks.A549-CMC was established according to the process of"co-incubation of YFSJP extract/control solution with A549→ elution of non-specific binding components→dissociation of binding components→LC-MS/MS identification of binding components".The components of YFSJP extract bound to A549 membrane were obtained by comparing the differences in dissociation solutions between the treatment group,control group and the last washing solution through LC-MS/MS.5.Pharmacodynamic verification of binding components in vitroCCK8 assay,apoptosis assay and cell cycle assay were used to detect the effects of binding components on the proliferation,apoptosis and cycle of A549 cells.The components that could inhibit A549 proliferation,promote apoptosis and arrest cell cycle were considered to be the effective components of YFSJP extract against NSCLC.6.Exploration of the membrane targets and mechanisms of YFSJP extract against NSCLC based on network pharmacologyVenn diagram was drawn by taking the intersection of NSCLC disease targets and YFS JP targets(that is,binding component targets),and the intersection part was the potential effect target of YFSJP against NSCLC.Kyoto Encyclopedia of Genes and Genomics(KEGG)pathway analysis and Gene ontology(GO)function annotation were performed on potential effective targets.And protein to protein intersection(PPI)network was constructied.The correlation between membrane proteins in intersection proteins and NSCLC was clarified by literature review,and the effect of membrane proteins on the prognosis of NSCLC patients was analyzed by Kaplan-Meier Plotter website.Finally,core membrane proteins and core pathways were selected out.The expression of core membrane protein on the membrane of A549 cells was detected by western blot.The binding ability of binding components with core membrane protein was verified by molecular docking.7.Verification of the effect of peimisine on the migration and invasion of A549 cells by targeting TGFBR2Peimisine,the binding component with the strongest binding ability to the core membrane protein TGFBR2,was selected for experiments in vitro.The binding between peimisine and TGFBR2 was further confirmed by molecular dynamics simulation and surface plasmon resonance(SPR).Molecular dynamics simulations analyzed the skeleton fluctuation analysis,which includes root-mean-square deviation(RMSD)of protein and ligand,root-mean-square fluctuation(RMSF)of protein,radius of Gyration(Rg)and binding force analysis.SPR experiment mainly offerred the kinetic parameters of the binding of peimisine and TGFBR2.Wound healing assay,transwell migration assay and transwell invasion assay were used to explore the effects of different concentrations of peimisine on the metastasis of A549 cells.Western blot was used to detect the epithelial-mesenchymal transition(EMT)related markers including E-cadherin,N-cadherin,Vimentin,Slug and TGFβpathway marker p-Smad2 and Smad2.Finally,TGFBR2 agonist TGFβ1 and TGFBR2 inhibitor SB-431542 were used to further verify the effect of peimisine on TGFβ pathway and related phenotypes.8.Effects of YFSJP and peimisine on A549-luc metastasis model(tail vein)Firstly,the A549-luc cell line was established by lentivirus transfection,and the bioluminescence detection in vitro was performed to confirm the successful establishment of the cell line.Then,the metastasis model by injecting A549-luc cells through tail vein was established,and the effects of YFSJP,peimisine and SB-431542 on the model were observed.Safety indicators including body weight,serum liver and kidney function,viscera index were evaluated.The efficacy was evaluated by monitoring the vivo imaging of the mice,observing the lung surface metastatic nodes and lung hematoxylin&eosin staining.Western blot was used to detect the expression of EMT-related markers and TGFβ pathway markers.Results:1.Chemical analysis of YFSJP extract73 chemical components in the YFSJP extract were identified by UPLC-Q-TOF-MS/MS.2.Establishment of HPLC fingerprint of YFSJP extract and content determination of the four componentsThe chromatographic conditions were as follows:chromatographic column:Techway JADE-PAK 3.5 μm ODS-AQ(150×4.6 mm);Mobile phase:acetonitrile-0.1%phosphoric acid water;Column temperature:30℃;Detection time:115 minutes;Injection volume:10 μL;Detection wavelength:210 nm;Flow rate:1.0 mL/min;Elution gradient of mobile phase:5%-17%acetonitrile 0-41 min,17%-17%acetonitrile 41-51 min,17%-25%acetonitrile 51-75 min,25%-30%acetonitrile 75-85 min,30%-34%acetonitrile 85-105 min,34%-40%acetonitrile 105-110 min and 40%-50%acetonitrile 110-115 min.20 common peaks were obtained by comparing 10 different batches of samples.The similarity of HPLC chromatogram of different batches of samples was above 0.9,suggesting that the fingerprint method could be used for the quality control of the YFSJP extract.Eight peaks were identified by comparison with the standard,among which peak 2 was chlorogenic acid,peak 5 was isofraxidin,peak 8 was astilbin,peak 12 was dactylorhin A,peak 14 was rosaminic acid,peak 15 was militarine,peak 16 was ginsenoside Re,peak 17 was ginsenoside Rb1.The methods for the content determination of the four components were successfully established,and the methods had good specificity,precision,stability,repeatability and sample recovery.The linear fitting equations of the four components were:y=1.3624x-0.1302(isofraxidin),y=0.4436x-0.0177(rosaminic acid),y=0.1655x+0.0051(militarine),y=0.0236x-0.0085(ginsenoside Re).In 10 batches of YFSJP extract,the concentrations of isofraxidin,rosmarinic acid,militarine and ginsenoside Re were 49.314～83.203 μg/mL,80.046～258.135μg/mL,113.955～334.982 and 1010.275～1627.119 μg/mL respectively.3.Establishment of pretreatment method for cell dissociation solution samplesConsidering the recovery rate and the convenience of operation,HLB column-eluent A was finally selected as the final sample pretreatment method for CMC.The specific steps were as follows:loading 4 mL methanol and 4 mL ultrapure water for activation and equilibrium→ loading of 1 mL YFSJP extract at a flow rate of 1 mL/min,standing for 1 min→leaching with 5 mL ultrapure water→elution with 50%acetonitrile aqueous solution→nitrogen blowing and constant-volume process of the elution solution.4.Establishment of A549-cell membrance chromatography(CMC)and identification of binding componentsA549-CMC was successfully established.40 mg/mL YFSJP extract was used for the cubation in CMC.After five time of washing,no obvious integrable chromatographic peak was found in the fifth washing solution.Therefore,the number of washing for A549-CMC was determined to be five times.LC-MS/MS screened and identified nine A549 membrane binding components in the YFSJP extract:isofraxidin-7-o-β-glucoside,peimisine,peimine,militarine,ginsenoside Rt5,ginsenoside Rbl,ganoderic acid A,ginsenoside Rc,ginsenoside Ro.5.Pharmacodynamic verification of binding components in vitro.Molecular biology experiments showed that ginsenoside Rb1,ginsenoside Rc,ginsenoside Ro,peimisine and peimine could inhibit the proliferation of A549 cells,ginsenoside Ro,peimisine and peimine could arrest the A549 cell cycle,and peimisine and peimine could promote the A549 apoptosis.The above five components were the effective components of YFSJP extract against NSCLC.6.Exploration of the membrane targets and mechanisms of YFSJP extract against NSCLC based on network pharmacologyNetwork pharmacology analysis enrolled 35 intersection targets between YFSJP and NSCLC.The results of KEGG analysis enriched 7 pathways,most of which were closely related to cancer,such as pathways in cancer,prostate cancer,TGFβ signaling pathway,transcriptional misregulation in cancer,and proteoglycans in cancer.GO functional annotation results suggested that the intersection targets were mainly enriched in molecular functions such as protein kinase activity,nuclear receptor activity,histone kinase activity,endopeptidas activity,protein kinase binding,transmembrane receptor protein kinase activity.They were mainly enriched in biological processes such as protein phosphorylation,cellular response to lipids,response to nutritional levels,branching morphogenesis of epithelial tubes,and regulation of kinase activity.They mainly located in sites associated with biological membranes such as secretory granule lumen,endoplasmic reticulum lumen,membrane rafts,and serine/threonine protein kinase complexes.Subsequently,the PPI network of intersection targets was successfully constructed.These results suggest that YFSJP may play its pharmacologic action against NSCLC through multiple targets and multiple pathways.Among the membrane targets in intersection targets,TGFBR2,KDR and HSP90AA1 are related to the occurrence and development of NSCLC,and are closely related to the prognosis of NSCLC.HSP90AA1 and KDR are protective factors for lung adenocarcinoma,TGFBR2 is a risk factor for lung adenocarcinoma and a protective factor for lung squamous cell carcinoma.TGFBR2 ranked the highest in the association number of compounds and pathways,that is,4 compounds and 5 pathways were associated with TGFBR2.These results suggest that TGFBR2 may be the core membrane target of YFSJP and TGFβ pathway may be the core pathway for suppressing NSCLC.Molecular docking results showed that libdock scores of TGFBR2(receptor)and 9 binding components(ligand)were all greater than 100,indicating that the ligands and receptors had good binding ability.Among them,peimisine had the highest binding score with the ATP-pocket in TGFBR2,and it was selected for further verification.7.Verification of the effect of peimisine on the migration and invasion of A549 cells by targeting TGFBR2The RMSD of the protein basically reached stabilization after 10 ns,whereas the ligand reached its first equivalent state after 5 ns simulation and its second equivalent state after 50 ns simulation.The Rg of the protein-ligand complex remained stable throughout the simulation,suggesting protein-ligand complex binding was stable.There are four main regions with high RMSF values,including key residues K277,H328,N332 and D397 in the TGFBR2 catalytic pocket,indicating that residues within the catalytic pocket have high flexibility during the simulation.It possibly attributed to the interaction between the receptor and ligand.Binding force analysis was performed using the last 10 ns of the entire simulation,and R339,N332,L386 and E335 showed strong binding to their ligands.However,residues V250,K252,V258,V276,L305,A326,K330,G331,W336,L386 and V387 also contributed to the binding of protein to ligands to a lesser extent.In contrast,residues such as A326,F327,H328,A329,H340,and C396 have a negative effect on protein-ligand binding.The above results suggest that four key residues of the catalytic pocket dominate the binding of peimisine to TGFBR2,which may inhibit TGFBR2 function through hydrophobic interaction force.SPR results showed that 20-160 μM concentrations of peimisine had a high response value in a dose-dependent manner when flowing through the sensor chip.It suggested that peimisine has a strong affinity with TGFBR2.The kinetic parameters of the binding between peimisine and TGFBR2 were obtained by kinetic fitting:ka=1.40e2(1/(M*s)),kd=1.00e-2(1/s),KD=7.15e-5(M).Scratch test and transwell migration and invasion results suggested that 25,50 and 100 μM concentrations of peimisine can inhibit the migration and invasion of A549 cells in a dose-dependent manner.Western blot results showed that they can inhibit EMT and TGFβ pathways,which was reflected in the up-regulation of E-cadherin and down-regulation of N-cadherin,Vimentin,Slug and p-Smad2/Smad2.The subsequent agonist/inhibitor application in forward and reverse validation showed that the agonist TGFβ1 can reverse the effect of peimsine on regulating TGFβ pathway to inhibit EMT and anti-metastasis,and the inhibitor SB-431542 could promote the effect of peimisine.8.Effects of YFSJP and peimisine on A549-luc metastasis model(tail vein)A certain bioluminescence signal could be detected when A549-luc cells was more than 1250,and the signal intensity increased in response to the increasing number of cells.It suggested that A549-luc cells were successfully constructed.The results of animal experiments suggested that the body weight of each treatment group was heavier than that of the model group,but there was no statistical significance.There were no significant differences in the organ indexes of heart,liver,spleen,lung and kidney among the groups.Concentration of the serum alanine aminotransferase,aspartate aminotransferase,and creatinine were not significantly different among the groups,and all were within normal reference ranges.The serum urea nitrogen(BUN)in the model group was very low.And the BUN in each treatment group was in the normal range or slightly below the normal range,which was significantly higher than that in the model group,especially in the YFSJ and SB-431542 groups.The results of bioluminescence monitoring suggested that metastases began to be observed after 4 w of drug administration,and the extent of metastases in each treatment group was significantly less than that in the model group.The bioluminescence signal of the last imaging in each treatment group was significantly lower than that in the model group,but there was no statistical significance.Compared with the model group,the number of metastatic nodules on the lung surface in each treatment group was reduced,and the SB-431542 group was the least.And there was no significant difference among YFSJP,peimisine i.g.,and peimisine i.p.groups.The results of hematoxylin-eosin staining of the left lung supported the above result.The results of western blot showed that YFSJP,peimisine i.g.,peimisine i.p.and SB-431542 could inhibit TGFβ pathway and EMT to varying degrees.Conclusions:Taken together,cell extraction/UPLC-MS/MS,network pharmacology,and molecular biology-based analysis comprise a feasible strategy to explore active ingredients in YFSJP,which provides a new insight into study the effective ingredients in Chinese formula.1.The method of quality control of YFSJP extract and CMC were establilshed.Nine components binding to A549 membrane were analyzed and identified:isofraxidin-7-O-β-glucoside,peimisine,peimine,militarine,ginsenoside Rt5,ginsenoside Rb1,ganoderic acid A,ginsenoside Rc,and ginsenoside Ro.Among them,ginsenoside Rbl,ginsenoside Rc,ginsenoside Ro,peimisine and peimine can inhibit A549 cells and are the effective components of YFSJP against NSCLC.2.YFSJP exerted anti-NSCLC effects through multiple targets and multiple pathways,among which TGFBR2 was the core target and TGFβ pathway was the core pathway.Peimisine,one of the effective components in YFSJP can target TGFBR2 to regulate TGFβ pathway to inhibit EMT,and finally anti-NSCLC metastasis.3.YFSJP and its effective component peimisine could inhibit the metastasis of A549-luc metastasis model,and had good safety in vivo and improved hypo-azotemia.The underlying mechanism is related to inhibit TGFβ pathway and thus inhibit EMT. |
PDF Full Text Request