Molecular Mechanisms Of Stellera Chameajasme L.and Its Flavonoid Active Ingredient Chamaejasmenin B In The Treatment Of Glioblastoma Cell Lines

1.Objective1.1 To research the potential chemical ingredients,key targets,signaling pathways of Stellera Chameajasme L.(RXLD)acting on Glioblastoma(GBM),network pharmacology,molecular docking,and biological verification methods were exploited.1.2 In vitro experiments were carried out to study the molecular mechanisms of the regulated cell death(RCD)of GBM cells induced by the RXLD flavonoid constituent Chamaejasmin B(CHB),and to demonstrate the correlation between different RCD mechanisms and their roles in cell survival and death.1.3 Explore the distributions of differentially expressed genes(DEGs)that CHB acting on GBM in different signaling pathways,screen the biological functions,core targets and signaling pathways the DEGs regulated,as well as obtain the docking activity between CHB and core targets,by using the methods of transcriptomics and molecular docking.2.Methods2.1 The chemical structures of the compounds extracted from RXLD were explored by literature search.The 3D structures of the chemical constituents were constructed by PubChem database and Chemdraw softerware and then imported into the PharmMapper database to obtain the pharmacophore models.2.2 The gene targets of GBM were obtained from Genecard,OMIM,Genebank and other databases.The pharmacophore models-targets network and protein-protein interaction(PPI)network were constructed by String database and visualized by Cytoscape.2.3 Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes pathway(KEGG)enrichment were conducted by Bioconductor software.The relationship of pathways and candidate genes were visualized by Cytoscape to screen the key target genes.2.4 Pymol,Autodock,and Vina software were applied to acquire the molecular docking results.2.5 We explored CCK-8 assay,Annexin V-FITC/PI assay,Annexin V-PI assay,cell scratch healing assay,transwell cell migration assay,DCF staining ROS assay,and JC-1 staining mitochondrial membrane potential to verify the anti-GBM function of RXLD extracts and the effects of apoptosis,autophagy and ferroptosis which CHB induced in GBM.The expression of core targets in the related pathways was detected by Western Blot assay.2.6 The destiny of cells survival or death,in the condition of fetal bovine serum(FBS)or hungry,was detected after CHB combined with rapamycin(Rapa)or 3-Methyladenine(3-MA).2.7 The expression levels of LC3A/B,Bax,caspase-8,caspase-3,NRF2,p62,NCOA4,Ferritin was detected using WB methods when the GBM cell lines were treated by CHB combination with Rapa,3-MA,Ferrostatin-1(Fer-1),Erastin,and Deferoxamine mesylate(DFO).2.8 The expression of p-AKT,p-mTOR,p-MEK,p-ERK was dectected to verify the signaling pathways regulated by CHB acting on GBM.2.9 We applied transcriptomic sequencing(RNA-Seq)to obtain the differentially expressed genes(DEGs),constructed protein interaction(PPI)network of DEGs by String platform and CytoNCA software,performed the KEGG enrichment analysis by Bioconductor,and visualized the target-KEGG network to obtain the core targets and signaling pathways.The molecular docking was carried out by using Pymol,autodock,and vina software to perform molecular docking ability between key targets and CHB.3.Results3.1 We explored 744 genes in pharmacophore models in which 216 potential genes were related with GBM.The essential components in RXLD were Neochamaejasmin A,Wikstrol A,Isochamaejasmin,Chamaejasmine,and Subtoxin A.3.2 GO enrichment showed that oxidative stress,cell proliferation,cell cycle,cell invasion and migration were involved in the biological processes.The crucial pathway was MAPK pathway in which HRAS,PRKCB,MAPK9,CCND1,and TP53 were core targets.3.3 In total of 7 chemical ingredients in RXLD have strong spontaneous docking activities with MAPK9.3.4 RXLD extracts exerted anti-GBM functions by promoting apoptosis via P53 pathway,arresting the cell cycle in G2/M and S phases,inhibiting cell migration via Wnt/β-Catenin pathway,and regulating oxidative stress and ferroptosis through p62/NRF2.3.5 CHB could inhibit the cell viability of GBM cells in a time-concentration-dependent manner,and induce cell apoptosis at an early stage.3.6 CHB could induce A172 cells to undergo imbalanced autophagic flux and NCOA4-mediated ferritinophagy with the characteristc of LC3A/B-Ⅱ activation and p62 accumulation.3.7 Autophagy played cell protect mechinsms in FBS condition,while menifested an inhibition effect in the starvation condition.The dysregulated autophagic flux and ferritinophagy were co-regulated cell apoptosis and oxidative stress response:the accumulation of p62 was more dependent on the activation of the ferroptosis,while the oxidative stress and apoptosis were more related to the activation of autophagic flux.3.8 The levels of p-AKT and p-mTOR were gradually decreased from 0 to 12 hours,while the levels of p-MEK and p-ERK were significantly increased from 0.5 to 2 hours after CHB acting on GBM.3.9 In total of 1729 DEGs were obtained by RNS-Seq.The target-KEGG network screened out the ITGA,ITGB,PIK3R1,PI3K/AKT signaling pathway and MAPK signaling pathway as the core targets and signaling pathways.Molecular docking shown that CHB had strong docking activity with ITGA,ITGB and PIK3R1.4.Conclusion4.1 The RAS/MAPK pathway might be the upstream pathway for RXLD to exert its anti-GBM effect,and further regulate the P53 pathway,Wnt/β-Catenin pathway,oxidative stress and ferroptosis pathways.4.2 CHB might be a PIK3R1 inhibitor.On the one hand,CHB induced ferritinophagy and activated autophagic flux by inhibiting AKT subsequently activating MEK/ERK pathway.On the other hand,CHB inhibited autophagic flux by suppression of PI3 K/AKT/mTOR pathway.The interaction and influence of the two machinsms co-determined the dysregulation of autophagic flux with the effect of LC3A/B-Ⅱ activation and p62 accumulation.