| Objective Combined with network pharmacology,molecular docking technology and multi-public database mining,through in vivo and in vitro experiments and verification,the effects and mechanisms of B.rynchopetera and its combination with cisplatin on lung adenocarcinoma were studied,in order to provide a theoretical basis for the clinical drug development of B.rynchopetera.Methods The molecular targets and mechanism of B.rynchopetera against non-small cell lung cancer were analyzed by network pharmacology and molecular docking.Twenty Sprague-Dawley male rats were intragastrically administered with B.rynchopetera decoction according to the conversion dose of human and rat body surface area,and the B.rynchopetera-containing serum was prepared according to the serum pharmacological method.The effects of B.rynchopetera and its combination with cisplatin on the growth,proliferation,migration and invasion of non-small cell lung cancer A549 cells were studied by CCK-8,cell scratch,Transwell chamber and Western blot assays.In this experiment,48 C57BL/6 Lewis non-small cell lung cancer mice were randomly divided into 4 groups(n = 12)according to the average tumor size : model control group,B.rynchopetera group,cisplatin group and drug combination group(B.rynchopetera + cisplatin).The body weight,long diameter and short diameter of the tumor of C57BL/6 non-small cell lung cancer mice were recorded.The changes of body weight and tumor growth of C57BL/6 non-small cell lung cancer mice were observed and recorded.After treatment,the tumor tissue,spleen and thymus organs were weighed,and the growth inhibition rate and immune organ index of solid tumor were calculated.HE staining and IHC staining were performed on the tumor tissues of each group to observe the tissue morphology of each group and the positive expression of the target protein in each group.Through network pharmacology and molecular docking,the molecular mechanism of action of B.rynchopetera against lung adenocarcinoma was analyzed.The effects of B.rynchopetera on the differential gene expression and prognosis of lung adenocarcinoma were analyzed by TCGA and GTEx databases.Through the public databases such as NCBI,Gene Cards and KEGG,the ferroptosis mechanism of B.rynchopetera against lung adenocarcinoma and its enhanced sensitivity to cisplatin was analyzed.Results(1)Through network pharmacology analysis,there were 11 potential hub targets of B.rynchopetera against non-small cell lung cancer : ALB,HSP90AA1,SRC,CASP3,MAPK1,AR,CDC42,PGR,NOS3,CTSB,GSTP1.The results of Gene Ontology(GO)enrichment analysis showed that the biological processes were mainly related to the negative regulation of vascular endothelial growth factor receptor signaling pathway,Fcγreceptor signaling pathway and exogenous apoptosis signaling pathway.The cell components mainly involved cytoplasm,plasma membrane,membrane pits,protein complexes,nuclei,exosomes,and cytoplasm.Molecular functions mainly involved protein binding,Rho-GDP dissociation inhibitor binding,same protein binding,mitogen-activated protein kinase binding,and enzyme binding(all P < 0.05).Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the items related to non-small cell lung cancer were : VEGF signaling pathway,Estrogen signaling pathway,Adheren Junction signaling pathway,Rap1 signaling pathway and MAPK signaling pathway(all P < 0.05).Molecular docking results showed that the hub compounds had good binding activity with putative hub targets.(2)In vitro cell experiments,the results of CCK-8 showed that compared with the blank control group,the cell proliferation of the B.rynchopetera-containing serum group,cisplatin group and drug combination group decreased significantly.Compared with the cisplatin group,the cell proliferation in the drug combination group was significantly decreased(all P < 0.05).The results of migration and invasion experiments showed that compared with the blank control group,the cell scratch healing area and the number of cell migration and invasion in the B.rynchopetera-containing serum group,cisplatin group and drug combination group decreased.Compared with the cisplatin group,the cell scratch healing area and the number of cell migration and invasion in the drug combination group decreased(all P < 0.05).The results of Western blot showed that compared with the blank control group,the expression of VEGF,SRC,CDC42 and Bcl-2 protein was down-regulated,and the expression of CASP3 and Bax protein was increased in the B.rynchopetera-containing serum group,cisplatin group and drug combination group(all P < 0.05).(3)In vivo animal experiments,there was no significant difference in body weight changes between the B.rynchopetera group and the model control group(P > 0.05);compared with the model control group and the B.rynchopetera group,the body weight of mice in the cisplatin group and the drug combination group decreased(P < 0.05).In tumor growth,the subcutaneous tumor of the model control group grew fastest,and the subcutaneous tumor of the drug combination group grew slowest.In terms of immune organ index,compared with the model control group,the immune organ indexes of the B.rynchopetera group,the cisplatin group and the durg combination group decreased(all P < 0.05).In terms of tumor growth inhibition rate,compared with the model control group,the tumor weight of the B.rynchopetera group,the cisplatin group and the drug combination group decreased,and the tumor inhibition rate was higher than that of the model control group.Compared with the cisplatin group,the tumor weight of the drug combination group was reduced,and the tumor inhibition rate was higher than that of the cisplatin group(P< 0.05).The results of hematoxylin-eosin(HE)staining of tumor tissue showed that the tumor nucleus of the model control group was large and round,and the nuclear-cytoplasmic ratio was high.The tumor cells in each treatment group showed more nuclear condensation,hyperchromatosis or fragmentation,marginalization,and vacuolization.Pathological nuclear division was significantly reduced.The results of immunohistochemical(IHC)staining of tumor tissue showed that compared with the model control group,the positive expression of VEGF,SRC,CDC42 and Bcl-2 proteins in the B.rynchopetera group,cisplatin group and drug combination group was higher,and the protein expression of CASP3 and Bax was lower.(4)There are 13 potential hub targets for the treatment of lung adenocarcinoma by B.rynchopetera through network pharmacology analysis : SPHK1,PDGFRB,CXCR4,MAPK1,CCNE1,NFE2L2,CDK5,HIF1 A,GSK3B,PTGS2,MMP9,CDK1,STAT1.The results of gene ontology enrichment analysis showed that biological processes were mainly related to the positive regulation of smooth muscle cell proliferation,the positive regulation of protein entry into the nucleus,the negative regulation of apoptosis process,the positive regulation of cell migration,protein phosphorylation,peptidylserine phosphorylation and peptidylthreonine phosphorylation.Cell components mainly involve centrosome,nucleoplasm,cyclin-dependent protein kinase holoenzyme complex,nucleus and cytoplasm.Molecular functions mainly involved kinase activity,protein binding,ATP binding,enzyme binding,p53 binding and other molecular functions(all P < 0.05).Encyclopedia of Genomes enrichment analysis showed that the items related to lung adenocarcinoma were : cancer pathway,micro RNA cancer pathway,IL-17 signaling pathway,chemokine signaling pathway,PI3K-Akt signaling pathway,VEGF signaling pathway,EGFR tyrosine kinase inhibitor resistance,etc.(all P < 0.05).Molecular docking results showed that the hub compounds had good binding activity with putative hub targets.(5)There are significant differences in the expression of CCNE1,CDK1,MMP9,STAT1,SPHK1 and PDGFRB between lung adenocarcinoma tumor tissues and normal tissues(all P < 0.05).The effects of CCNE1,CDK1,PTGS2,GSK3 B,CXCR4,HIF1 A and SPHK1 on the overall survival of lung adenocarcinoma were different(all P < 0.05).The putative hub targets,SPHK1,PDGFRB,MAPK1,NFE2L2,HIF1 A,GSK3B and PTGS2,of B.rynchopetera in the treatment of lung adenocarcinoma is related to ferroptosis.Conclusion(1)B.rynchopetera can act on VEGF,SRC,CDC42,CASP3,Bcl-2,Bax and other targets,regulate VEGF signaling pathway,inhibit the growth,proliferation,migration and invasion of human lung adenocarcinoma A549 cells,and inhibit the proliferation of mouse lung adenocarcinoma Lewis cells;(2)B.rynchopetera may act on SPHK1,PDGFRB,CXCR4,MAPK1,CCNE1,NFE2L2,CDK5,HIF1 A,GSK3B,PTGS2,MMP9,CDK1,STAT1 targets to play an anti-lung adenocarcinoma role;(3)B.rynchopetera may act on CCNE1,CDK1,SPHK1 targets to affect the survival and prognosis of lung adenocarcinoma;(4)B.rynchopetera may act on ferroptosis-related genes such as SPHK1,PDGFRB,MAPK1,NFE2L2,HIF1 A,GSK3B,and PTGS2 to enhance the sensitivity of cisplatin against lung adenocarcinoma. |
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