Exploring The Mechanism Of Inhibiting Peritoneal Angiogenesis By Fushen Recipe Based On Dll4/Notch1 Signaling Pathway

Objective:1.Observation of the effect of Fushen Recipe on peritoneal capillary endothelial cell angiogenesis model in human2.Exploring the effect and mechanism of action of intervention of peritoneal angiogenesis with Fushen RecipeMethods:1.Peritoneal capillary endothelial cell angiogenesis model constructionHuman peritoneal capillary endothelial cells were used as the target,and the peritoneal capillary endothelial cells were stimulated with the appropriate VEGF concentration through CCK-8 screening to construct a peritoneal angiogenesis model,and endothelial cell migration and tube-lumen formation ability were observed through cell wound healing assay and tube-lumen formation assay,and the expression of VEGF and Ang2,the relevant indexes of angiogenesis,were detected by WB to comprehensively evaluate the peritoneal capillary endothelial cell angiogenesis model.2.Transcriptomic study on the intervention of peritoneal angiogenesis by Fushen RecipeBased on the successful construction of a peritoneal angiogenesis model,the drug-containing serum of Fushen Recipe was used to intervene in peritoneal angiogenesis.The mi RNA transcriptome sequencing and bioinformatics analysis were used to further explore the potential mechanisms of peritoneal endothelial cell angiogenesis and Fushen Recipe’s intervention in peritoneal angiogenesis.3.Study on the mechanism of inhibiting peritoneal angiogenesis by Fushen RecipeFirstly,we evaluated the efficacy of Fushen Recipe in inhibiting peritoneal angiogenesis by angiogenesis assays such as arterial vascular ring and PCR and WB assays.Subsequently,the m RNA and protein expression levels of Dll4,Notch1,RBP-J,MAML1,Hes1 and Hey1,the factors related to Dll4/Notch1 signaling pathway screened by GO and KEGG enrichment analysis,were detected by PCR and WB techniques to elucidate the mechanism of action of Fushen Recipe in inhibiting peritoneal angiogenesis.Results:1.Peritoneal capillary endothelial cell angiogenesis model construction(1)Optimal concentration of VEGF screening: 80ng/ml.When VEGF at 80 ng/ml was used to intervene in peritoneal capillary endothelial cells,their cell viability showed the best state,with a significant increase in proliferation capacity compared to the blank group without VEGF stimulation(P<0.001).(2)Evaluation of peritoneal angiogenesis model: The results of cell wound healing assay and tube lumen formation assay showed that the model group with the concentration of80ng/ml VEGF intervention could significantly stimulate the migration of human peritoneal capillary endothelial cells and tube lumen formation compared with the blank group,as quantified by Image J software,the area of cell wound healing in the model group increased significantly(P<0.01),and the number of nodes and branches of tube lumen in the model group increased significantly(P<0.01,P<0.05).WB results showed that the protein expression of VEGF and Ang-2,proteins related to angiogenesis,was increased in the model group compared with the blank group(P<0.05).2.Transcriptomic study on the intervention of peritoneal angiogenesis by Fushen Recipe(1)Screening of the best concentration of Fushen Recipe drug-containing serum: Fushen Recipe high concentration(3.058g/ml),compared with the model group,when Fushen Recipe drug-containing serum concentration was high,had the best inhibition rate on peritoneal vascular endothelial cell angiogenesis model(P<0.01).(2)mi RNA transcriptome sequencing results and target gene prediction: 59 differentially expressed mi RNAs were screened in the blank group compared with the model group(P<0.05),including 31 up-regulated mi RNAs and 28 down-regulated mi RNAs.83 differentially expressed mi RNAs were screened in the model group compared with the Fushen Recipe group(P<0.05),including 42 up-regulated mi RNAs and 41 down-regulated mi RNAs.The target genes were predicted using Target Scan(v5.0)and mi Randa database respectively for the mi RNAs with significant differences after comparison between two groups.814 target genes of differentially expressed mi RNAs were predicted in the blank and model groups,and 3116 target genes of differentially expressed mi RNAs were predicted in the model and dosing groups.(3)GO and KEGG analysis of target genes: In terms of the mechanism of peritoneal angiogenesis,the results of GO enrichment analysis showed that the biological processes involved in the differential mi RNA target genes mainly involved tubular epithelial cell morphology,etc.,the cellular components were mainly cell leading edge,etc.,and the molecular functions were mainly SMAD binding;the results of KEGG enrichment analysis mainly involved FOXO signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,etc.The results of KEGG enrichment analysis mainly involved FOXO signaling pathway,PI3K-Akt signaling pathway,MAPK and other signaling pathways.The results of GO enrichment analysis showed that the biological processes involved in the differential mi RNA target genes were mainly related to the regulation of cell development and protein ubiquitination,and the components of cells were mainly transcriptional regulatory complexes,and the molecular functions were mainly SMAD binding;the results of KEGG enrichment analysis mainly involved Notch signaling pathway,TGF-β signaling pathway and cellular senescence The results of KEGG enrichment analysis mainly involved Notch signaling pathway,TGF-β signaling pathway and cellular senescence pathway.3.Study on the mechanism of inhibiting peritoneal angiogenesis by Fushen Recipe(1)Evaluation of the efficacy of Fushen Recipe: The arterial vascular ring experiment showed that compared with the model group,the sprouting of microvessels in the arterial ring of rats treated with Fushen Recipe drug-containing serum group was not only inhibited,but also the new microvessels appeared apoptotic;the cell wound healing assay and tube formation experiment showed that compared with the VEGF model group,the migration ability of peritoneal capillary endothelial cells and the tube formation ability were significantly weakened after the optimal serum intervention of Fushen Recipe.The PCR results showed that the expression levels of VEGF and VEGFR2 m RNAs in peritoneal capillary endothelial cells decreased after the intervention of Fushen Recipe(P<0.05,P<0.01);WB results showed that compared with the model group,the expression levels of VEGF,VEGFR2,and Ang2 protein were decreased in the Fushen Recipe-containing serum group(P<0.05).(2)Exploring the mechanism of inhibiting peritoneal angiogenesis with Fushen Recipe:PCR results showed that compared with the model group,Dll4,Notch1,RBPJ m RNA expression levels were decreased in Fushen Recipe-containing serum group(P<0.05,P<0.01,P<0.01),and Hes1,MAML1,Hey1 m RNA levels only had a decreasing trend;WB results showed that compared with the model group,Dll4,Notch1,RBP-J,and Hes1 protein expression levels were decreased in the Fushen Recipe-containing serum group(P<0.05,P<0.01,P<0.01,P<0.05),and MAML1 protein levels only showed a decreasing trend.Conclusion:1.80 ng/ml VEGF stimulation of human peritoneal capillary endothelial cells could successfully construct a peritoneal angiogenesis model.2.Fushen Recipe inhibits peritoneal capillary endothelial cell proliferation,migration and tube formation,and improves peritoneal angiogenesis through multi-target and multi-pathway.3.The potential mechanism of inhibition of peritoneal angiogenesis by Fushen Recipe may be achieved through inhibition of the Dll4/Notch1 signaling pathway.