Research On The Effective Material Basis Of Chinese Medicine Caulis Spatholobi And The Mechanism Of Anti-cervical Cancer

Purpose:The literature indicates that Spatholobi Caulis flavonoids?SCF?and Spatholobi Caulis tannin?SCT?have a significant therapeutic effect on cervical cancer.Based on previous research,the main components of SCF and SCT were analyzed by mass spectrometry,and analysis of related components by Ultra Performance Liquid Chromatography?UPLC?.The pharmacodynamics and mechanism of SCF and SCT in HeLa cervical cancer cells were further studied.In this paper,a three-dimensional microfluidic chip was used to simulate the in vivo pharmacodynamic mechanism,and the appropriate administration time and dose were determined.Furthermore,in our experiments,SCF and SCT promoted apoptosis and inhibited proliferation in HeLa cells.The differentially expressed genes between cervical cancer patients and healthy subjects were screened by an analysis of The Cancer Genome Atlas?TCGA?and Gene Expression Omnibus?GEO?databases.Through a bioinformatics analysis of related genes combined with molecular docking technology,related genes and proteins that can interact with SCF and SCT were predicted.The regulatory effect of SCF and SCT on related pathogenic circular RNAs?circRNAs?was determined by RT-PCR.The above experiments suggest that SCF and SCT can inhibit proliferation and promote apoptosis in HeLa cells by acting on related circRNAs in HeLa cells.Therefore,SCF and SCT can treat cervical cancer through precise targeting.Material and method:1.The analysis was performed using an Agilent 6550 iFunnel Q-TOF mass spectrometer.The compounds were identified through an analysis of an online database and published articles.The Fingerprint of SCF and SCT.Content detection was established by the method of Ultra Performance Liquid Chromatography?UPLC?.2.In this experiment,a chip model was fabricated using soft lithography.SU8-2075 negative photoresist was spin coated onto a clean silicon plate and photolithographically bonded to the design mask.After a programmed baking process,a chip model was fabricated.Polydimethylsiloxane?PDMS?and a hardener were mixed at a ratio of 15:1 and cast to form a fluid channel layer?FCL?and a substrate layer.The PDMS and hardener were mixed at a ratio of 8:1 and poured to form a gas channel layer?GCL?.The FCL and GCL were trimmed and perforated,and the layers were oxidized with ultraclean glass in oxygen plasma for 3 minutes and chemically bonded to form a chip3.A cell suspension containing sodium alginate and medium containing calcium chloride were injected into the chip through a peristaltic pump,and a 3D cell culture environment was allowed to form inside the chip.The cell cluster growth was observed after 3,6,9,and 12days of culture.A culture solution containing sodium citrate was injected into the 3D chip by a peristaltic syringe pump.The colloid was dissolved,and the cells were collected for the preparation of cell smears.The cell pellet was stained with Wright-Giemsa staining reagent to facilitate the observation of the state of the cell mass.4.The different doses of SCF,SCT and positive drugs were administered after 12 days of cell culture.After few hours,the cells were lysed in sodium citrate solution by a peristaltic syringe pump,and the cell pellet was collected and made into a cell smear.The fluorescent dyes calcein?AM?and propidium iodide?PI?were used for staining,and the cells were incubated with the prepared working solution at room temperature.The prepared cell smear was observed under an inverted fluorescence microscope.The cell viability?%?was calculated using Image-Pro Plus?IPP?software.5.Flow cytometry analysis of cell apoptosis and the cell cycle.Low,medium and high concentrations of SCF,SCT and the positive control drug were added.After 36 hours,the percentage of apoptotic cells was determined using an Annexin V-FITC Apoptosis Detection kit.Apoptosis was analyzed using flow cytometry in conjunction with FlowJo software.The effect of SCFs on the cell cycle was assessed using a cell cycle assay kit.Then,flow cytometry was used in combination with ModFit software to analyze the changes in the cell cycle.6.The differentially expressed genes between normal subjects and cervical cancer patients were analyzed using the TCGA and GEO databases.The genes with equal expression were integrated using R software and subjected to a KEGG analysis by DAVID.The GeneMANIA application in Cytoscape software was used to analyze the gene interactions and core genes in related pathways.The relationship between proteins related to the core gene was analyzed by STRING,and the target protein was also analyzed.7.SCF and SCT can interact with amino acids in proteins via forces,such as hydrogen bonding and?-?conjugation.The common nucleus of these flavonoid structures was screened,and the mother nucleus structure was used as a representative structure of SCF and SCT in the molecular docking analysis.The molecular modeling and docking studies were performed using the AutoDock tool.We designed and studied the affinity of compounds to related target proteins through molecular docking studies.8.A TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit and a TransStart Top Green qPCR SuperMix kit were used.The designed back-to-back primers were combined with cDNA to amplify the circRNAs.The RT-PCR data were analyzed using the 2^-??CTmethod.RegRNA was used to analyze miRNAs that target circRNAs.Results:1.8 different batches of SCF of UPLC fingerprints was created,the SCF of similarity evaluation analysis showed that the SCF of different batches of the best compatibility with the reference fingerprint atlas compared to the similarity was different.Fourteen compounds were initially identified in SCF.The contents of 4 kinds of monomers was detected.The content of Ononin in 3.73 mg·g^-1,Formononetin content in 1.63 mg·g^-1,Liquiritin content in 5.33 mg·g^-1,Daidzin content in 4.64 mg·g^-1.2.8 different batches of SCT of UPLC fingerprints was created,the SCT of similarity evaluation analysis showed that the SCT of different batches of the best compatibility with the reference fingerprint atlas compared to the similarity was different.Fifteen compounds were initially identified in SCT.The contents of 3 kinds of monomers was detected.The content of Epigallocatechin in 2.26 mg·g^-1,Ethyl gallate content in 0.97 mg·g^-1,Epicatechin gallate content in 1.22 mg·g^-1.3.The results of the designed chip application prove that the chip not only promotes the formation of a cancer cell mass but also provides a basis for the therapeutic effect of the drug on cancer in the human body.As the time increased,the number of cells and the size of the cell mass increased.After culturing the cells for 12 days,the cell mass grew to 140?m.Thus,the 3D microfluidic chip designed in this experiment can simulate the growth of cell clusters in the human body and provides a basis for screening drug doses and delivery times.4.Through the efficacy of SCF in the 3D microfluidic chip,SCF can effectively inhibit cell proliferation and reduce the cell cluster size.The results of the drug intervention in the chip showed that SCF can dissociate and disperse cell clusters by inhibiting cell-cell interactions.In addition,as the dose increased,the survival rate of the cells significantly decreased,showing a clear dependence on the efficacy and measurement.The best actuation duration is36 hours.The optimum dose were 0.50 mg·mL^-1?1.00 mg·mL^-11 and 2.00 mg·mL^-1.5.The apoptosis analysis showed that the SCF significantly promoted cell apoptosis.The percentage of cells in both early and late apoptosis significantly increased with increasing doses.The results of the cell cycle analysis of the drug intervention were analyzed.These results demonstrate that SCF arrest HeLa cells in the S phase and inhibit cell proliferation by inhibiting the synthesis of intracellular DNA.SCT arrest HeLa cells in the G₀/G₁ phase6.Based on the bioinformatics analysis,Based on the gene interaction score results,54 genes with higher scores were screened.A multi-corresponding protein interaction network analysis revealed that the network contained 54 hub proteins.The selected proteins can provide a basis for the identification of genes and proteins targeted by SCF and SCT.7.The docking analysis results indicated that SCF can bind the proteins like FANCB protein,MYBL2 protein,PRKACB protein and PTTG1 protein.SCT can bind the proteins like ATR protein,BLM protein,E2F3 protein and FANCB protein.Conclusion:1.In this study,the fingerprints and components of SCF and SCT were obtained.On this basis,the content of multi-index components was detected.2.The 3D microfluidic chip can be used for cell cluster culture and drug screening research.3.SCF and SCT have an effect on inducing cell apoptosis and inhabiting cell division within a certain concentration range.4.The selected proteins can provide a basis for the identification of genes and proteins targeted by SCF and SCT.5.Screening of genes and proteins that may play a role in SCF and SCT by molecular docking.6.SCF and SCT are predicted to exert their effects by affecting the interaction between relevant miRNAs and circRNAs.In summary,SCF and SCT can inhibit the growth of HeLa cells by regulating relevant circRNAs.